Protein-protein interactions in mono- and bi-partite geminiviruses: functional analysis and use of aptamers for plant protection
Zusammenfassung der Projektergebnisse
Geminiviruses are a family of plant DNA viruses characterized by their circular, singlestranded genomes encapsidated within small twinned icosahedral particles from which the group derives its name. They are important pathogens of crop plants in the Middle East. Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted monopartite geminivirus, causes extensive damage to tomatoes, whereas Watermelon chlorotic stunt virus (WmCSV), a bipartite member of the family and recently discovered in Israel and Palestine, threatens watermelon and melon production. Another bipartite geminivirus, the Indian cassava mosaic virus (ICMV) was included as a well studied model for the bipartite group. One of the critical determinants of infection is a multifaceted interaction between the various viral proteins. In addition, viral suppressors of the plant silencing machinery are known to contribute to the severity of the disease. In this project, we have studied the various molecular aspects of the viruses protein-protein interactions and cellular localization involved in disease development. We have developed means to interfere with viral protein-protein interactions needed for the completion of viral life-cycle and for intra- and intercellular movement. The following major goals of this project were: 1) Characterization of protein-protein interactions between TYLCV CP and PCP proteins in vivo and in vitro. 2) Use of combinatorial peptide libraries and the yeast two hybrid system (Y2H) as well as the reverse two hybrid system for identification of high-affinity peptides that disrupt TYLCV CP-CP and CP-PCP interactions and anti-NLS peptides that bind the TYLCV CP NLS. 3) Generation of transgenic tomato and Nicotiana benthamiana plants expressing high-affinity inhibitory peptides and anti-NLS peptides, and study their resistance to viral infection. 4) Cloning of WmCSV genes coding for the NSP and MP proteins, and identify their putative nuclear localisation and export sequences, as well as the cellular targeting domains within the WmCSV NSP, PCP, and MP. 5) Testing the multitude of transport proteins of ICMV (PCP, CP, NSP and MP) with respect to their binding capacities for viral protein partners as well as to aptamers derived from the screening described above, in order to decide whether the desired blocking of intra- and intercellular transport is specific to TYLCV and WmCSV or applicable for other geminiviruses as well. 6) Selection of inhibitory peptides that bind to TYLCV and ICMV proteins which may interfere with their functions. To this aim, the respective proteins were expressed in plants and fission yeasts, purified, and interesting candidate peptides were selected which may be used in transgenic plants to be tested for their function against TYLCV, WmCSV and ICMV in future projects. High resolution localization of the proteins by fluorescence microscopy and electron microscopy yielded indications where in the cell the protein-protein interference has to be engineered. These accomplishments illustrate the productive collaboration between our laboratories, based on complementary expertise and efficient exchange of ideas, information, and research tools and know-how.
Projektbezogene Publikationen (Auswahl)
- (2009). Geminiviruses. In "Torque Teno Virus: The Still Elusive Human Pathogens" (H. zur Hausen, de Villiers, E.-M., Ed.), Current Topics in Microbiology and Immunology, Vol. 331, pp. 185-226. Springer, Berlin
Jeske, H.
- (2010). First Report of Squash leaf curl virus (SLCV) on squash (Cucurbita pepo L.), melon (Cucumis melo L. ), and cucumber (Cucumis sativa L.) in the Northern West Bank of the Palestinian Authority. Plant Disease 94: (5): 640 (abstract)
Ali-Shtayeh, M. S., Jamous, R. M., Hussein, E. Y., & Alkhader, M.
- (2011) Effect of a single amino acid substitution in the NLS domain of Tomato yellow leaf curl virus-Israel (TYLCV-IL) capsid protein (CP) on its activity and on the virus life cycle. Virus Res. 158: 8- 11
Yaakov, N., Levy, Y., Belausov, E., Gaba, V., Lapidot, M. and Gafni, Y.
- (2012). First Report of watermelon chlorotic stunt virus (WmCSV) in watermelon (Citrullus lanatus), and cucumber (Cucumis sativa) in the Palestinian Authority. Plant Dis., 96, 1, pp 149
Ali-Shtayeh, M. S., Jamous, R. M., Hussein, E. Y., Mallah, O. B., Abu-Zeitoun, S. Y.
- (2013). In vivo self-assembly of TMV-like particles in yeast and bacteria for nanotechnological applications. J. Virol. Methods 189, 328-340
Kadri, A., Wege, C., and Jeske, H.
(Siehe online unter https://doi.org/10.1016/j.jviromet.2013.02.017) - (2013). Squash leaf curl virus (SLCV): a serious disease threatening cucurbits production in Palestine. Virus Genes. 11/2013
Ali-Shtayeh, M. S., Jamous, R. M., Hussein, E. Y., Mallah, O. B., Abu-Zeitoun, S. Y.
(Siehe online unter https://doi.org/10.1007/s11262-013-1012-1) - (2014). Molecular characterization of watermelon chlorotic stunt virus (WmCSV) : A Serious Disease Threatening Watermelon Production in Palestine. Viruses, 2014, 6(6), 2444-2462
Ali-Shtayeh, M. S., Jamous, R. M., Mallah, O. B., Abu-Zeitoun, S. Y.
(Siehe online unter https://doi.org/10.3390/v6062444)
