Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana

Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

305 TRANSGENIC CLONED PIGS EXPRESSING ORANGE FLUORESCENT PROTEIN KUSABIRA-ORANGE

Transgenic (Tg)-cloned pigs expressing fluorescent proteins are very useful in research such as cell or tissue transplantation studies. In this study, we produced Tg-cloned pigs with an orange fluorescent protein, humanized Kusabira-Orange (huKO), and analyzed the characteristics of these pigs. Fetal fibroblast cells transduced with huKO gene by a gene silencing-resistant retroviral vector, pDΔNhuKO (Suzuki et al. 2002 J. Neurochem. 82, 953–960) were used as nuclear donors. Recipient cytoplasts were prepared using oocytes matured in vitro in NCSU23. The nuclear transfer (NT) embryos were transferred into oviducts of estrus-synchronized recipient gilts after culture in PZM-5 for 1 or 2 days. Cloned pigs obtained were analyzed by Southern blotting for the transgene integration. A total of 23 organs and tissues, including brain, eye, internal and reproductive organs, skin, skeletal muscle, and cartilage were examined by fluorescence stereomicroscopy. Cryo- and paraffin-embedded tissue sections were also prepared to examine fluorescence expression. Immunofluorescent staining of brain cryosection and flow cytometry analysis of peripheral blood cells were performed to identify huKO-expressing cells. Transfer of 429 NT embryos into 4 recipients resulted in 18 (4.2%) cloned offspring. Southern blotting analysis of the cloned pigs confirmed transduction of 2 to 17 copies of the huKO gene in each pig. Autopsy was performed in 6 pigs, and orange fluorescence was confirmed in all the tissues and organs examined in each pig. In addition, prominent orange fluorescence was detected in pancreatic islets and renal glomeruli, indicating that these transgenic pigs are ideal for islet cell transplantation studies. Expression of huKO was also detected in neurons, microglia, and astrocytes in the brain, and granulocytes, monocytes, macrophages, lymphocytes, and platelets in the peripheral blood cells, whereas the expression level in red blood cells was lower. Re-cloning was performed using primary culture fibroblast cells established from 2 Tg-cloned pigs. Transfer of 300 re-cloned embryos into 4 recipients resulted in 3 pregnancies. A re-cloned fetus at Day 44 of gestation showed systemic fluorescence. These results demonstrate that the retroviral vector pD?NhuKO is resistant to gene silencing in pigs, that transduction and expression of the huKO gene had no lethal effects on fetal development, and that a Tg-cloned pig expressing orange fluorescence can be produced by NT of fetal fibroblast cells transduced with the huKO gene. This study was supported by PROBRAIN.