SummaryGeminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation con®ning the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360 degrees without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3-4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

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Tamarillo, or tree tomato, is native to South America. We examined the in vitro antioxidant and anti‐inflammatory properties of ethanolic extracts of skins, seeds, pulp, and whole tamarillo fruit in an LPS (lipopolysaccharide) induced RAW 264.7 mouse macrophage cell model. Chemically, tamarillo skin exhibited the highest total phenol (116.75 ± 2.78 mg gallic acid/g tamarillo), Trolox® equivalent antioxidant capacity (TEAC) (67,018.7 ± 2,539.9 meq Trolox®/g tamarillo) and ORAC values (4,684.9 ± 267.5 μM Trolox®/100 ml). The highest anthocyanin values (17.86 ± 0.85 mg/g) were associated with the seeds. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assays were used to determine non‐toxic tamarillo extract doses and viability in RAW 264.7 cells. In the absence of LPS stimulation, tamarillo seeds and whole fruit, but not skin or pulp extracts, significantly induced NO formation (P < 0.05). In contrast, tamarillo skin, but not seeds, pulp, or whole tamarillo extracts significantly suppressed NO formation (P < 0.05) in the presence of LPS stimulation. These results suggest that the tamarillo fruit extracts modulate NO formation in the RAW 264.7 model and that these effects vary depending on the composition of the extract in question, such that seeds are net inducers of NO, while skins inhibit LPS‐induced NO formation.