2 resultados para TRANSCRIPTIONAL REGULATORS
em DRUM (Digital Repository at the University of Maryland)
Resumo:
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.
Resumo:
RNA is an underutilized target for drug discovery. Once thought to be a passive carrier of genetic information, RNA is now known to play a critical role in essentially all aspects of biology including signaling, gene regulation, catalysis, and retroviral infection. It is now well-established that RNA does not exist as a single static structure, but instead populates an ensemble of energetic minima along a free-energy landscape. Knowledge of this structural landscape has become an important goal for understanding its diverse biological functions. In this case, NMR spectroscopy has emerged as an important player in the characterization of RNA structural ensembles, with solution-state techniques accounting for almost half of deposited RNA structures in the PDB, yet the rate of RNA structure publication has been stagnant over the past decade. Several bottlenecks limit the pace of RNA structure determination by NMR: the high cost of isotopic labeling, tedious and ambiguous resonance assignment methods, and a limited database of RNA optimized pulse programs. We have addressed some of these challenges to NMR characterization of RNA structure with applications to various RNA-drug targets. These approaches will increasingly become integral to designing new therapeutics targeting RNA.