Tomato bushy stunt virus (TBSV) provides a good model system to investigate
molecular virus-host interactions in plants. P22 and P19 proteins encoded by TBSV
contribute to multiple invasion-associated functions. Green fluorescence-mediated
visualization of TBSV invasion in this study suggests that virus exit from inoculated
epidermal cells is a crucial event. Close examination of one P22 mutant showed that it
had lost the capacity to move between epidermis and mesophyll which was possibly due
to an altered subcellular localization. P19 is a potent suppressor of RNA interference
(RNAi) in various systems by forming dimers that bind 21-nucleotide (nt) duplex siRNAs
(short interfering RNAs), to affect the programming of the RNA-induced silencing
complex (RISC). P19 is attractive for biotechnological and research purposes to prevent
RNAi of certain value-added genes in plants. To obtain a good plant-based expression
platform, a suppression-active mutant P19 was expressed in transgenic N. benthamiana
lines. This is the first example of P19 accumulating to detectable levels in a transgenic
plant and initial results suggest it is actively suppressing RNAi. Furthermore, to
investigate the correlation between siRNA binding of P19 and its various biological roles,
predicted siRNA-interacting sites of TBSV P19 were modified, and the corresponding TBSV mutants were used to inoculate plants. Substitutions on siRNA-contact sites on the
central domain of P19 resulted in more severe symptoms in N. benthamiana compared to
those affecting peripheral regions. All tested combinations of siRNA-binding mutations
were associated with reduced accumulation of total TBSV-derived siRNAs, and loss of
siRNA sequestration by P19. Additionally, some modifications were found to cause
RNAi-mediated disappearance of viral and host materials in N. benthamiana but not in
spinach. In conclusion, exit out of epidermal cells is a key host range determinant for
TBSV and particular amino acids on P22 may influence this by regulating the proper
subcellular localization. Mutant P19 transgenic plants were successfully established with
minor physiological effects to be applied as a platform to study RNAi and to over-express
proteins. Finally, a compromised P19-siRNA binding impacts symptom development,
systemic invasion, integrity of virus plus host RNA and proteins, and that all in a hostdependent
manner.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2008-08-33 |
| Date | 16 January 2010 |
| Creators | Hsieh, Yi-Cheng |
| Contributors | Scholthof, Herman B. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation |
| Format | application/pdf |
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