Abstract
Citrus tristeza virus (CTV) is one of the most devastating pathogens of citrus. Its genome is organized into 12 open reading frames (ORFs), of which ten ORFs located at the 3′-terminus of the genome have multiple biological functions. The ten genes at the 3′-terminus of the genome of a severe isolate (CTV-S4) and three ORFs (CP, CPm and p20) of three other isolates (N4, S45 and HB1) were cloned into pGBKT7 and pGADT7 yeast shuttle vectors. Yeast two-hybridization (Y2H) assays results revealed a strong self-interaction for CP and p20, and a unique interaction between the CPm of CTV-S4 (severe) and CP of CTV-N4 (mild) isolates. Bimolecular fluorescence complementation also confirmed these interactions. Analysis of the deletion mutants delineated the domains of CP and p20 self-interaction. Furthermore, the domains responsible for CP and p20 self-interactions were mapped at the CP amino acids sites 41–84 and p20 amino acids sites 1–21 by Y2H. This study provided new information on CTV protein interactions which will help for further understanding the biological functions.
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Acknowledgments
This work was financially supported by National Natural Science Foundation of China (Grant No. 312721) and Chinese Ministry of Agriculture, Industry Technology Research project (Grant No. 201203076). The authors would like to thank assistant professor Xiaofeng Wang, Virginia Polytechnic Institute and State University, USA, for critical revisions of this manuscript.
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11262_2014_1100_MOESM3_ESM.tif
Fig. S1. Mapped region required for CP self-interaction by yeast two-hybrid assay. Full-length CP amino acid sequence and truncated mutants CPs were fused to BD, and AD vectors individually, equal volume of prey and bait were mixed and used to transformed S. cerevisiae strain Y2HGold. The transformed cells were cultured on medium and high selective media. Schematic representation of truncated CPs CP cloned into yeast shuttle vector used to test for interaction (left). Interacting pairs grew and turn blue on selective SD/QDO/X/AbA plates, and the LacZ activity was tested using β-galactosidase filter lift assay and liquid assay using ONPG with unit recorded as “Millers unit” (right).(TIFF 759 kb)
11262_2014_1100_MOESM4_ESM.tif
Fig. S2. Mapped domain of p20-p20 interacting domain. The p20 and truncated p20 fragments encoded by BD-type and AD-type plasmids used in each experiment are shown in the left columns. For galactosidase colony lift assay and quantification test by ONPG, the transformed cells were streaked onto synthetic medium and high selective media (SD-DDO/X/AbA and QDO/X/AbA) followed by incubation at 30°C for three days. The two right columns show the ability to grow on high selective media QDO/X/AbA and β-galactosidase activity with ONPG measured in “Miller’s unit” (right) (TIFF 890 kb)
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Nchongboh, C.G., Wu, Gw., Hong, N. et al. Protein–protein interactions between proteins of Citrus tristeza virus isolates. Virus Genes 49, 456–465 (2014). https://doi.org/10.1007/s11262-014-1100-x
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DOI: https://doi.org/10.1007/s11262-014-1100-x