CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.

Bat-plant pollination interactions in southern Thailand

The overwhelming majority of flowering plant species depend on animals for pollination, and such pollinators are important for the reproductive success of many economically and environmentally important plant species. Yet pollinators in the Old World tropics are relatively understudied, particularly paleotropical nectarivorous bats (Pteropodidae), and much is unknown about their interactions with night-blooming plant species. To better understand these bat-plant pollination interactions, I conducted fieldwork in southern Thailand for a total of 20 months, spread across three years. I examined the foraging times of pteropodid bat species (Chapter 1), and found that strictly nectarivorous species foraged earlier, and for a shorter duration, than primarily frugivorous species. I also studied year-long foraging patterns of pteropodid bats to determine how different species track floral resources across seasons (Chapter 2). Larger species capable of flying long distances switched diets seasonally to forage on the most abundant floral species, while smaller species foraged throughout the year on nearby plant species that were low-rewarding but highly reliable. To determine which pteropodid species are potentially important pollinators, I quantified the frequency and effectiveness of their visits to six common bat-pollinated plant taxa for an entire year (Chapter 3). The three strictly nectarivorous species were responsible for almost all pollination, but pollinator importance of each bat species varied across plant species. I further examined the long-term reliability of these pollinators (Chapter 4), and found that pollinator importance values were consistent across the three study years. Lastly, I explored mechanisms that reduce interspecific pollen transfer among bat-pollinated plants, despite having shared pollinators. Using a flight cage experiment, I demonstrated that these plant species deposit pollen on different areas of the bat’s body (mechanical partitioning), resulting in greater pollen transfer between conspecific flowers than heterospecific flowers (Chapter 5). Additionally, while I observed ecological and phenological overlap among flowering plant species, pollinators exhibited high floral constancy within a night, resulting in strong ethological separation (Chapter 6). Collectively, these findings illustrate the importance of understudied Old World bat pollinators within a mixed agricultural-forest system, and their strong, interdependent interactions with bat-pollinated plant species within a night, across seasons, and across years.

Dynamics of a large submersed plant bed in upper Chesapeake Bay

A large SAV bed in upper Chesapeake Bay has experienced several abrupt shifts over the past half-century, beginning with near-complete loss after a record-breaking flood in 1972, followed by an unexpected, rapid resurgence in the early 2000’s, then partial decline in 2011 following another major flood event. Together, these trends and events provide a unique opportunity to study a recovering SAV ecosystem from several different perspectives. First, I analyzed and synthesized existing time series datasets to make inferences about what factors prompted the recovery. Next, I analyzed existing datasets, together with field samples and a simple hydrodynamic model to investigate mechanisms of SAV bed loss and resilience to storm events. Finally, I conducted field deployments and experiments to explore how the bed affects internal physical and biogeochemical processes and what implications those effects have for the dynamics of the system. I found that modest reductions in nutrient loading, coupled with several consecutive dry years likely facilitated the SAV resurgence. Furthermore, positive feedback processes may have played a role in the sudden nature of the recovery because they could have reinforced the state of the bed before and after the abrupt shift. I also found that scour and poor water clarity associated with sediment deposition during the 2011 flood event were mechanisms of plant loss. However, interactions between the bed, water flow, and waves served as mechanisms of resilience because these processes created favorable growing conditions (i.e., clear water, low flow velocities) in the inner core of the bed. Finally, I found that that interactions between physical and biogeochemical processes led to low nutrient concentrations inside the bed relative to outside the bed, which created conditions that precluded algal growth and reinforced vascular plant dominance. This work demonstrates that positive feedbacks play a central role in SAV resilience to both chronic eutrophication as well as acute storm events. Furthermore, I show that analysis of long-term ecological monitoring data, together with field measurements and experiments, can be an effective approach for understanding the mechanisms underlying ecosystem dynamics.