Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion.

Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.

Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells.

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments--such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen--that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H(+)-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.

Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants.

The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants.