Analysis of the interaction between Tomato torrado virus proteins using the yeast two-hybrid system
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Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute, Władysława Węgorka 20, 60-318 Poznań, Poland
Aleksandra Obrępalska-Stęplowska
Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute, Władysława Węgorka 20, 60-318 Poznań, Poland
Submission date: 2013-10-22
Acceptance date: 2013-10-30
Journal of Plant Protection Research 2013;53(4):416–423
Ten years ago for the first time the new picorna-like virus species – Tomato torrado virus (ToTV) – was found and described on tomato plants. The isolates of this pathogen were reported in Europe, America, and Oceania including Australia. Because of its unique biological and molecular features, ToTV was classified to the new genus Torradovirus, in the Secoviridae family. In Poland, three isolates: Wal’03, Kra, and Ros ToTV were identified on greenhouse tomato cultivars. At present, the biology and the genome structure of this virus are characterised. But there is no data extending beyond the bioinformatics analyses about the function of viral proteins, polyproteins, and non-coding sequences, as well as possible interactions between viral, host and vector factors that may be important for the infection process, encapsidation, transport in plants, and transmission. In this study, we have undertaken a search for the possible protein-protein interaction of ToTV encoded proteins using the yeast two-hybrid (Y2H) system. The viral genome fragments covering full sequences for nine known proteins of ToTV were amplified using specific primers with characteristic recombination sites. This process enabled the construction of basic entry clones for each protein that further facilitated manipulations with prepared constructs using Gateway technology. Two-hybrid assays were performed in the yeast strain and tested interactions of ToTV proteins were analysed in several combinations using auxotrophy markers. Our analyses did not reveal the presence of interactions between ToTV domains. Surprisingly, no interactions were found in the case of various CP subunits as well as between CP subunits and 3A protein, that in some virus families are known to play a role in viral life cycle. This role includes virion assembly or cell-to-cell transport. The lack of interactions may be a result of the limitation of this experimental system, or suggest that these proteins may interact indirectly, or require the presence of genomic RNAs or some host factors.
The authors have declared that no conflict of interests exist.
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