RT Dissertation/Thesis
T1 Analysis of the interaction between the helper component proteinase (HC-Pro) of Zucchini yellow mosaic virus (ZYMV) and the plant RNA methyltransferase Hua enhancer 1 (HEN1)
A1 Jamoos,Rana
WP 2012/08/24
AB The helper component-proteinase (HC-Pro) is a multifunctional protein found among potyviruses. It plays multiple roles in the viral infection cycle and some of these functions have been mapped to different regions of the protein. The subcellular localization of several viral RNA silencing suppressor (RSS) proteins was identified. In this study, we have shown that the Zucchini yellow mosaic virus (ZYMV) HC-Pro wild type (HC-ProFRNK) and its mutant, HC-ProFINK, had a diffuse cytoplasmic localization and formed aggregates along the endoplasmatic reticulum (ER).
HC-ProFRNK and HC-ProFINK were stably expressed in N. benthamiana and A. thaliana plants. In addition, the HC-ProFRNK and HC-ProFINK were fused to a nuclear localization signal (NLS) sequence (NLS-HC-ProFRNK and NLS-HC-ProFINK) and these transgenes constructs were also stably transformed into N. benthamiana and A. thaliana. Expression of all four transgenes caused different effects in the two plant species. HC-ProFRNK?producing A. thaliana plants displayed severe phenotypic alterations. In A. thaliana, the HC-ProFINK led to a reduced number of seed set. In N. benthamiana expressing HC-ProFRNK/FINK, generally no or only slight phenotypic changes were monitored. The NLS-HC-ProFRNK/FINK-producing plants displayed clear phenotypes. Flower malformations and severe reduction of seed set were the most conspicuous observations made. In general, more severe developmental disturbances were observed in transgenic A. thaliana than in N. benthamiana plants.
ZYMV HC-Pro RSS activity was previously demonstrated in N. benthamiana plants by transient expression experiments. In this study, RSS activity was confirmed in N. benthamiana lines stably expressing ZYMV HC-ProFRNK/FINK. Notably, these plants did not show significant morphological alterations. Because the RSS activity of HC-Pro leads to enhanced transgene expression, our ?symptom-free? transgenic N. benthamiana plants may serve as a platform for over-expression of foreign genes.
In tobacco, transient or over-expression of rgs-CaM mimicked the phenotypic effects of Tobacco etch virus (TEV) HC-Pro, indicating that TEV HC-Pro may up-regulate rgs-CaM expression. However, our data revealed no significant difference in the levels of rgs?CaM mRNA in N. benthamiana plants expressing HC-ProFRNK/FINK when compared with the steady-state mRNA level found in the wild type plants. It is likely that RSS proteins from related viruses do not necessarily exhibit identical effects on RNA silencing. In addition, plant species might also differentially respond to identical RSS proteins.
The small RNA (sRNA) binding activity of HC-Pro was evident in N. benthamiana plants co-expressing the HC-ProFRNK and an infectious transgene construct of Potato spindle tuber viroid (PSTVd). In comparison to PSTVd-infected N. benthamiana plants, Northern blot analysis showed increase accumulation of viroid-derived sRNAs in the double transformed plants.
	There is indirect evidence showing that in plants, transient or stable expression of HC-Pro results in decreased accumulation of methylated sRNAs. In this study, we demonstrated that recombinant ZYMV HC-Pro inhibited the methyltransferase activity of the A. thaliana Hua enhancer 1 (AtHEN1) in vitro. Moreover, we found that HC-ProFINK lacking sRNA-binding activity, also inhibited AtHEN1 activity. In contrast, truncated HC-Pro and total soluble bacterial proteins did not affect AtHEN1 activity. Using enzyme-linked immunosorbent assays, we provided evidence that the HC-ProFRNK/FINK, both bound to AtHEN1. Our results strongly indicated that inhibition of the AtHEN1 activity by HC-Pro is probably due to direct interactions between both proteins. We concluded that AtHEN1 inhibition and sRNA-binding activities of HC-Pro are independent of each other. Using the yeast two-hybrid (Y2H) system, we could show that in contrast to RSS proteins from some other viruses, the HC-ProFRNK/FINK proteins did not interact with the Argonaute 1 (AGO1) protein.
Similar to previous reports our data confirmed that HC-Pro interacts with itself to form homodimers. Notably, only HC-ProFRNK but not HC-ProFINK was able to interact with itself. The conserved FRNK box is located in the central domain of HC-Pro and this domain has been previously shown to be involved in self-interaction.
It should be noted that parts of the work have been published in:
- Jamous R. M., Boonrod K., Fuellgrabe M. W., Ali-Shtayeh M. S., Krczal G. and Wassenegger M. (2011). The HC-Pro of the Zucchini Yellow Mosaic Virus (ZYMV) inhibits HEN1 methytransferase activity in vitro. J. Gen. Virol. 92, 2222-2226.
- Fuellgrabe M., Boonrod K., Jamous R., Moser M., Shiboleth Y., Krczal G. and Wassenegger M. (2011). Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro. Protein Expr. Purif. 75: 40-45.

K1 ZYMV 
PP Hohenheim
PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
UL http://opus.uni-hohenheim.de/volltexte/2012/755