Submitted to: Microscopy and Microanalysis
Publication Type: Proceedings
Publication Acceptance Date: 8/1/2002
Publication Date: 8/5/2002
Citation: Gallegos, L., Fambrough, K., Lartey, R.T., Ghoshroy, S. 2002. Inhibition of turnip vein clearing virus movement in seeds of infected arabidopsis thaliana plant; a microscopic study. Microscopy and Microanalysis. 8(Suppl. 2):1598-1599. Interpretive Summary: Systemic movement of virus infection in plants is important for disease development. After infection the virus moves from cell-to-cell until they reach the vascular bundles from where they spread to other parts of the plant to cause disease. Turnip vein-clearing virus (TVCV) causes systemic infection in Arabidopsis thaliana but unlike some plant viruses, it is not seed transmissible. In this study, we use infection of Arabidopsis by TVCV as a model system to study none seed transmission of viruses in plants. Arabidopsis plants were grown from seeds and mechanically infected with dilute TVCV virion suspension before emergence of the flowering stalks. Eight days after infection, lower uninoculated rosette and upper cauline leaves, flower and seed pods were examined by SDS-PAGE to detect the presence of coat protein (CP), as an indicator for successful systemic infection. Sections of flower samples were prepared and examined with Zeiss Axioplan microscope. Other samples were prepared and ultrathin sections were examined under a Hitachi H7000 transmission electron microscope. Post-section immunolabeling was performed with anti-TVCV CP antibody followed by colloidal gold conjugated secondary antibody. Viral coat protein was detected in whole seed pod and whole flower of the infected Arabidopsis. The confocal images did not show virus particles in the seeds of infected flower but were present in the ovary walls and all other parts of the flower. TEM images of longitudinal section of the flowers showed the anatomy of ovary and the distribution of virus particles as revealed by the immunofluorescence study. Furthermore, TEM images of infected tissues showed heavy thickening of the innermost cell layer of the ovary facing the seeds as opposed to a much thinner seed wall. No virus particles were observed in the seeds which was further confirmed by immunoelectron microscopy. The thick innermost cell layer of the ovary appears to have blocked movement of virus into the seeds resulting in development of virus free seeds. Future study will be carried out to identify host factors which may be involved in the blocking virus movement in seeds.
Technical Abstract: Viral movement in host plants is an important factor for development of disease. Following initial infection, plant viruses move through plasmodesmata (cell junctions in plants) to spread cell-to-cell until they reach vasculature.1 Viruses then move systemically through the vasculature and establish disease. Over the past decade, Arabidopsis thaliana has emerged as a model system to study various plant processes and plant-microbe interactions. Turnip vein clearing virus (TVCV), a tobamovirus has recently been shown to infect Arabidopsis.2 Although the virus infects vegetative and reproductive tissues, its apparent inability to spread to seeds prevents seed transmission of TVCV. In this study, we investigated the inhibition of viral movement in mature reproductive structures of Arabidopsis thaliana using light, confocal laser scanning, and transmission electron microscopy. Arabidopsis thaliana (ecotype Columbia) plants were grown from seeds. Before the emergence of the flowering stalk, two rosette leaves per plant were mechanically inoculated using a dilute TVCV virion suspension. After the 8th day of inoculation, lower uninoculated rosette leaf, upper cauline leaf, flower and seed pods were analyzed by SDS-polyacrylamide gel to detect the presence of coat protein (CP), an indicator for presence of virus. Intact flower samples from systemically infected plants were fixed, dehydrated, and embedded in paraffin and Spurr’s epoxy resin. The paraffin embedded samples were sectioned longitudinally and immunolabeling was performed using anti-TVCV CP antibody followed by Cy5-conjugated secondary antibody. Thick longitudinal sections were made from the resin embedded samples and photographed in a Zeiss Axioplan microscope. Same blocks were used to generate ultrathin longitudinal sections and observed under a Hitachi H7000 TEM. Post-section immunolabeling was performed using anti-TVCV CP antibody followed by 12nm colloidal gold conjugated secondary antibody. Infected Arabidopsis plants showed presence of viral coat protein in whole seedpod and whole flower. Later, plants grown from seeds of infected plants showed total absence of virus infection indicating absence of seed transmission. The confocal image shows absence of virus particles in the seeds of infected flower and heavy presence of virus in ovary wall and all other areas of the flower. Thick longitudinal sections of flower and a low magnification TEM image show the anatomy of ovary and the distribution of virus particles as revealed by the immunofluorescence study. The TEM images show a heavy thickening of the innermost cell layer of the ovary facing the seeds as opposed to a much thinner seed wall. No virus particles were observed in the seed and it was further confirmed by immunoelectron microscopy. The thick innermost cell layer of the ovary seems to block movement of virus into the seeds resulting in development of virus free seeds. However, the actual cause of inhibition of viral movement beyond the ovary wall is yet to be understood. Future study will be carried out to identify a potential host factor, if any blocking virus movement in seeds.