The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.

BackgroundThe genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).ResultsFollowing inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production.ConclusionsThis study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants.

Many reports have shown the beneficial effects of consumption of pine seeds and pine seed oil. However, few studies have examined the biological effect of pinolenic acid (PNA; 5,9,12-18:3), the main fatty acid in pine seed oil. In this study, using murine macrophage RAW264.7 cells as a model, we examined the effect of PNA on polyunsaturated fatty acid (PUFA) metabolism, prostaglandin (PG) biosynthesis and cyclooxygenase-2 (COX-2) expression. Results showed that PNA was readily taken up, incorporated and elongated to form eicosatrienoic acid (ETrA, 7,11,14-20:3) in macrophage cells. A small portion of this elongated metabolite was further elongated to form 9,13,16-22:3. The degree of incorporation of PNA and its metabolites into cellular phospholipids varied with the length of incubation time and the concentration of PNA in the medium. Incubation of PNA also modified the fatty acid profile of phospholipids: the levels of 18- and 20-carbon PUFA were significantly decreased, whereas those of 22-carbon fatty acids increased. This finding suggests that PNA enhances the elongation of 20-carbon fatty acids to 22-carbon fatty acids. The syntheses of PGE(1) from dihomo-gamma-linolenic acid (DGLA, 8,11,14-20:4) and PGE(2) from arachidonic acid (ARA, 5,8,11,14-20:4) were also suppressed by the presence of PNA and its metabolite. As the expression of COX-2 was not suppressed, the inhibitory effect of PNA on PG activity was attributed in part to substrate competition between the PNA metabolite (i.e., 7,11,14-20:3) and DGLA (or ARA).

Feeding mice a diet deficient in n-3 fatty acids for three generations resulted in a 53% decrease in docosahexaenoic acid (DHA, 22:6n-3) in the brain. Maternal pup retrieval and social learning of a food preference are both tasks based on olfactory function. All dams made contact more readily with pups of their own dietary group, and animals of both dietary groups demonstrated the ability to learn a food preference through exposure to a conspecific that had previously eaten the food. Both groups showed similar ability to learn the location of the hidden platform in the Morris water maze, while the n-3 deficient animals were marginally faster in locating the platform on the cued trial. They were also more active when tested in the open field. While they did not differ in their duration of immobility in a forced swimming test, the deficient animals did have longer paw-lick latencies on a hot plate. Thus, in this study a significant reduction in brain n-3 fatty acid composition, while associated with some indications of change in emotional reactivity, did not impair olfactory function or learning of either a latent or spatial nature.

LXA4 may inhibit the progression of endometriosis possibly by anti-inflammation and anti-angiogenesis.