Comparative evaluation of resistance to potato virus Y (PVY) in three different RNAi-based transgenic potato plants.

Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.

Monitoring Virus Intercellular Movement from Primary Infected Cells to Neighboring Cells in Plants.

Viral cell-to-cell movement from the primary infected cells to neighboring cells is an essential step for viruses to establish systemic infection in plants. The classic experimental design for studying this process involves the application of a reporter protein such as ß-glucuronidase (GUS), green fluorescent protein (GFP), or monomeric red fluorescent protein (mRFP or mCherry). However, such experimental settings are unable to unambiguously distinguish primary and secondary infected cells. In recent years, we have developed several double-labeling potyvirus infectious clones. Upon introduction of such vectors into plant leaf tissues, primary infected cells emit dual fluorescence (green and red) whereas secondary infected cells emit only green fluorescence. In this chapter, we provide detailed protocols on (1) construction of a GFP and mCherry-tagged turnip mosaic virus infectious clone, (2) delivery of the recombinant viral clones into plant cells by agroinfiltration, (3) confocal imaging of viral cell-to-cell movement, and (4) analysis of viral systemic infection. Using this dual-color imaging system, we have revealed coat protein (CP) is essential for TuMV cell-to-cell movement. This system provides a valuable and robust tool to study plant virus cell-to-cell movement.

Interplay of HCPro and CP in the Regulation of Potato Virus A RNA Expression and Encapsidation.

Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity.

Detection of infectious bursal disease virus (IBDV) antibodies using chimeric plant virus-like particles.

The aim of the present study is to use Physalis mottle virus (PhMV) coat protein (CP) as a scaffold to display the neutralizing epitopes of Infectious bursal disease virus (IBDV) VP2. For this, three different chimeric constructs were synthesized by replacing the N-terminus of PhMV CP with tandem repeats of neutralizing epitopes of IBDV VP2 and expressed in Escherichia coli. Expression analysis revealed that all the three recombinant chimeric coat protein subunits are soluble in nature and self-assembled into virus-like particles (VLPs) as evidenced through sucrose density gradient ultracentrifugation. The chimeric VLPs were characterized by various biochemical and biophysical techniques and found that they are stable and structurally sound. When the chimeric VLPs were used as coating antigen, they were able to detect IBDV antibodies. These results indicated that the chimeric VLPs can be used as potential vaccine candidates for the control of IBDV, which needs to be further evaluated in animal models.

Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV.

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

Bottom-Up Assembly of TMV-Based Nucleoprotein Architectures on Solid Supports.

RNA-guided self-assembly of tobacco mosaic virus (TMV)-like nucleoprotein nanotubes is possible using 3'-terminally surface-linked scaffold RNAs containing the viral origin of assembly (OAS). In combination with TMV coat protein (CP) preparations, these scaffold RNAs can direct the growth of selectively addressable multivalent carrier particles directly at sites of interest on demand. Serving as adapter templates for the installation of functional molecules, they may promote an integration of active units into miniaturized technical devices, or enable their presentation on soft-matter nanotube systems at high surface densities advantageous for, for example, biodetection or purification applications. This chapter describes all procedures essential for the bottom-up fabrication of "nanostar" colloids with gold cores and multiple TMV-like arms, immobilized in a programmable manner by way of hybridization of the RNA scaffolds to oligodeoxynucleotides exposed on the gold beads.