In order to identify host factors which interact with the movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV), ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv. R12740-7A, which had previously been constructed by yeast two-hybrid sequencing transformation. The protein functions of the identified host factors were determined according to their gene annotations in GenBank. The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2H Gold. Sixty-nine interactor proteins were identified, which were divided into the following 10 classes according to their described functions: hydrolases; pathogenesis-related proteins; DNA binding proteins; phosphatases; ligases; proteins with catalytic activity; phenylalanine ammonialyases; peroxidases; NAD binding proteins; and proteins of unknown function. Bioinformatic analysis of gene homology suggested that phosphatases, pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host. This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.
Flexiviridae ; genetics ; metabolism ; Malus ; genetics ; metabolism ; virology ; Molecular Sequence Data ; Plant Diseases ; genetics ; virology ; Plant Proteins ; genetics ; metabolism ; Plant Viral Movement Proteins ; genetics ; metabolism ; Protein Binding ; Two-Hybrid System Techniques

Abstract:By using PVX derived vector pGR107, the effect of BYDV-MP nuclear localization signal on the movement of PVX was studied. BYDV-MP was cloned into pGR107 using GFP as an indicator. BYDV-MP was then shown to induce the systemic infection and exacerbate the symptom of PVX through infecting Nicotiana benthamiana. When the PVX gene encoding 25kD protein, which functioned as a systematic movemnet protein,was deleted and the above experiment was repeated, the result showed that BYDV-MP could compensate the systemic movement of PVX. A serial mutants with substitutions on the fifth, sixth and seventh amino acids of BYDV-MP nuclear localization signal was further constructed. It was found that the mutants at the fifth, sixth amino acids in BYDV-MP nuclear localization signal could only delay or weaken systemic movement of PVX whereas the mutant at seventh amino acid could entirely inhibit systemic movement of PVX.
Amino Acid Sequence ; Green Fluorescent Proteins ; genetics ; Luteovirus ; physiology ; Molecular Sequence Data ; Nuclear Localization Signals ; chemistry ; physiology ; Plant Viral Movement Proteins ; physiology ; Potexvirus ; genetics ; physiology

OBJECTIVETo test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana.

METHODSSeven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.

RESULTSThe 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression.

CONCLUSIONTransient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Plant Viruses ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Potexvirus ; genetics ; RNA Interference ; Tobacco ; genetics ; metabolism

For expression of foreign genes in plant, plant virus vector provides many advantages, such as high expression level, short expression period and wider plant hosts. In the present study, we report the expression of thymosin alpha 1 (Talpha1) in tomato fruits by potato virus X (PVX) vector. Talpha1 gene fragment from plasmid pGEM-T containing Talpha1 gene was cloned into plant virus vector pGR107 and the resulting pGR107-Talpha1 plasmid was confirmed by digestion with Sal I and Cla I. To express the Talpha1 protein, Agrobacterium tumefaciens GV3101 transformed with pGR107-Talpha1 was directly injected into tomato fruits through the fruit stylar apex at different developmental stages. The ELISA results showed that Talpha1 protein was expressed successfully in fruits, and the highest expression level was obtained from 2.5-3 week-old tomato fruits inoculated by bacterium at 1.0 OD600 density.
Agrobacterium tumefaciens ; genetics ; Fruit ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Genetic Vectors ; genetics ; Lycopersicon esculentum ; genetics ; metabolism ; Plants, Genetically Modified ; metabolism ; Potexvirus ; genetics ; metabolism ; Recombination, Genetic ; Thymosin ; analogs & derivatives ; biosynthesis ; genetics

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.
Animals ; Antibodies, Monoclonal/immunology* ; China ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay/methods* ; Female ; Hybridomas ; Solanum lycopersicum/virology* ; Mice ; Mice, Inbred BALB C ; Plant Diseases/virology* ; Potexvirus/metabolism* ; Sensitivity and Specificity ; Nicotiana