The Self Interaction Of TuMV HC-Pro And Its Interaction With Rieske Fe/S Protein Encoded By Arabidopsis Thaliana

Turnip mosaic virus (TuMV) is a definite member in the genus Potyvirus. TuMV naturally infects the plants belong to the Brassica family, but it can also infect and induce symptoms on Arabidopsis thaliana and Nicotiana benthamiana, which are now commonly used as the model system for study on the host-pathogen interaction. It has been reported that helper component-proteinase (HC-Pro) encoded by potyviruses is an important viral symptom determinant and a suppressor of RNA silencing. Previous studies had showed that HC-Pro can form dimer in-vitro and in infected plants, moreover the interaction of HC-Pro with plant cellular factors also had been reported. In this study, to gain better understanding on the molecular basis of HC-Pro function in the viral pathogencity, the interactions of TuMV (isolates UKl)-encoded HC-Pro with itself and an Arabidopsis host factor were investigated.Although there have been many studies reported the ability of HC-Pro to self interact, the yeast two hybrid (YTH) assay used to determine the HC-Pro domain required for its self-interaction showed somewhat conflicting results. Based on the previously reported mutagenesis studies on HC-Pro and sequence alignments of various HC-Pro of Potyviruses, five deletion mutants of HC-Pro were constructed and used to analysis the functional domain involved in its self-interaction. YTH and bimolecular fluorescence complementation (BiFC) assays were used to detect the self-interaction ability of HC-Pro and its deletion mutant derivatives. YTH and BiFC assays confirmed TuMV HC-Pro self interaction in yeast cells and plant cells or insect cells, respectively. Using YTH analysis, the central and C-terminal regions of HC-Pro were shown to be participated in the self-interactions, while in BiFC assay, all deletion mutants interacted with HC-Pro, including the N- terminal regions that did not show any interaction with HC-Pro in the YTH assay. The subcellular localization of TuMV HC-Pro was also studied by fluorescent fusion protein and was observed to be present in the cytoplasm and to form aggregates along the endoplasmic reticulum (ER). The over-expression of HC-Pro caused the morphological changes of the ER, which suggested that HC-Pro is associated with the ER.The previous work of our laboratory to screen for the potential host protein interactors with HC-Pro using the A. thaliana cDNA library has obtained several candidates protein interactors. One cDNA clone designated Pet C, which encodes the Rieske Fe/S protein was selected for further analysis. The sequence analysis of Rieske Fe/S protein suggested that it contains a single transmembrane helix, the N-terminal of mature protein includes a short loose amino acid sequence and the C-terminal hydrophilic domain provides the ligands for the [2 Fe-2S] cluster. Its interaction with HC-Pro was confirmed by using YTH and by BiFC in plant cells. Two truncated mutants of Pet C were then generated to determine the region required for binding of Rieske Fe/S protein to HC-Pro. The result showed that the mature protein of Rieske Fe/S protein ((56-229aa) is the binding site for HC-Pro, whereas the transit peptide is dispensable for the interaction with HC-Pro. Both the central (residues 101-300aa) and the C-terminal regions (residues 301-458aa) of HC-Pro can interact with the mature Rieske Fe/S protein, but the N-terminal region (residues 1-100aa), which is related to aphid transmission is dispensable for the interaction.To analysis whether the HC-Pro expression affects the subcellular localization of Rieske Fe/S protein, Rieske Fe/S was fused with GFP and transiently co-expressed with HC-Pro in plants. The observation of protein subcellular localization indicated that Rieske Fe/S:GFP protein accumulates in cytoplasm, partly forms aggregates, and especially punctuate spots and granular bodies around the chloroplast. Co-expression with HC-Pro did not change the subcellular localization of Rieske Fe/S protein significantly. However the expression of HC-Pro can enhance the expression of Rieske Fe/S, this may be because HC-Pro function as a viral silencing suppressor can increase the heterogeneous gene expression in plants.To analysis the function of Rieske Fe/S protein in plant, the cDNA fragment corresponding to Pet C gene of A. thaliana were inserted into Tobacco rattle virus (TRV) and Potato virus X (PVX) vectors and inoculated into Arabidopsis and N. benthamiana for virus-induced gene silencing (VIGS). The upper leaves of Arabidopsis and N. benthamiana systemically infected with TRV-Pet C or PVX-Pet C exhibited severe yellowing symptom. The plants become stunted and a substantial reduced of growth rate compared with TRV-or PVX-infected plants were observed. The analysis of chlorophyll fluorescence parameters revealed that the Fo values were increased, whereas the Fv/Fm values were decreased in TRV-Pet C-infected plants as compared with TRV-infected plants. Semi-quantitative RT-PCR result showed that the level of Pet C mRNA decreased about 30%-70% after VIGS. In TuMV-infected Arabidopsis, yellow mosaic and stunting are the most common symptoms, which maybe a consequence of the perturbation of the chloroplast structure and the degradation of chloroplast pigment. It is possible that the interaction of HC-Pro and Rieske Fe/S protein interferes with the import of the Rieske Fe/S protein into the chloroplast, so that the amount of Rieske Fe/S protein required for assembly of photosynthetic cytochrome b6/f complex could eventually be reduced, and in turn contribute to development of yellow mosaic symptom.