HC-Pro as a tool to study silencing suppressor-mediated induction of disease symptoms in plants.
Final Report Abstract
In plants, virus infection involves plant defense and virus counter defense mechanisms that act at the RNA level. In their life cycle, viruses produce double stranded RNA molecules (dsRNAs) that are processed into small interfering RNAs (siRNAs) by the plant RNA interference (RNAi) machinery. RNAi is associated with degradation of virus genomes through binding of siRNAs to complementary regions of viral RNAs. Viruses, in turn, are coding for so-called silencing suppressor (SS) proteins that interfere with RNAi at versatile processes. In this project, the SS function of the helper component proteinase (HC-Pro) of the Zucchini yellow mosaic virus (ZYMV) was investigated. HC-Pro proteins which are produced by all potyviruses bind small dsRNAs, including siRNAs and micro RNAs (miRNAs), thereby impeding RNAi. MiRNAs are vital regulators of gene expression that are transcribed from endogenous genes in all eukaryotes. Single amino acid replacements within the conserved FRNK motif of HC-Pro proteins can abrogate their sRNA binding activity. Infections with ZYMV strains producing sRNA binding-deficient HC-Pro proteins (ZYMV-AG strains) led to attenuated symptoms. We analyzed whether severe ZYMV-induced symptoms are associated with sequestration of miRNAs by HC-Pro. Indeed, in ZYMV-infected and noninfected or ZYMV-AG strains-infected squash plants, differential accumulation of miRNAs and their target gene transcripts indicated a direct correlation between miRNA malfunction and symptom expression. Analysis of recombinant ZYMV HC-Pro proteins, produced in bacteria and plants, confirmed that mutation of the FRNK motif can result in loss of sRNA binding. However, despite sRNA binding deficiency, FRNK mutants preserved SS activity. We identified a directed physical interaction between ZYMV HC-Pro and the plant-encoded sRNA- methylating enzyme, Hua enhancer 1 (HEN1). From these data we concluded that inhibition of methylation results in destabilization of sRNA which would account for a second HC-Pro SS activity. We provided indirect evidence for this hypothesis by generating transgenic plants stably expressing ZYMV HC-Pro. In nature, HC-Pro is active in the cytoplasm whereas miRNA methylation takes place in the nucleus. Targeting of sRNA-binding and sRNA- binding-deficient HC-Pro proteins into the nucleus led to symptom production in Nicotiana benthamiana and Arabidopsis thaliana. We propose that nuclear HC-Pro proteins impeded HEN1-mediated miRNA methylation which would account for the observed symptoms. Finally, a plant-encoded protein (QP) that enhanced the HC-Pro sRNA binding activity was identified, isolated and recombinantly expressed in bacteria. We are currently using the sRNA binding activity of HC-Pro/QP to the development a tool that can be employed for medical applications, e.g. miRNA diagnosis and monitoring progression of miRNA-mediated therapeutics.
Publications
- (2007). The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81, 13135-13148
Shiboleth Y.M., Haronsky E., Leibman D., Arazi T., Wassenegger M., Whitham S.A., Gaba V. and Gal-On A.
- (2011). Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro. Protein Expr Purif 75, 40-45
Füllgrabe M., Boonrod K., Jamous R., Moser M., Shiboleth Y., Krczal K. and Wassenegger M.
- (2011). The HC-Pro of the Zucchini Yellow Mosaic Virus (ZYMV) inhibits HEN1 methytransferase activity in vitro. J Gen Virol
Jamous R.M., Boonrod K., Fuellgrabe M.W., Ali-Shtayeh M.S., Krczal G. and Wassenegger M.
