| My studies used the geminivirus Cabbage leaf curl virus (CaLCuV) as a vector for viral-induced gene silencing (VIGS) of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis. Previous results showed that RBR is a key component of the cell cycle that controls entry into S-phase by inhibiting E2F transcription factors. Hyperphosphorylation of RBR by a cyclin-dependant kinase (CDK)/cyclin complex releases E2F which then upregulates genes required for DNA synthesis. Although RBR is constitutively expressed, even in non-dividing tissues, null mutations in RBR cause gametophytic lethality, and thus make VIGS an intriguing genetic approach. Using CaLCuV vectors to silence RBR in Arabidopsis we found a reduction of leaf expansion, a proliferation of meristem-like structures, and clusters of epidermal cell outgrowths, a significant reduction in leaf size and an increase in cell density, manifested as rounded clusters of unexpanded cells on the surface of leaves. There was an increase in meristem-like structures that retained some expression of the meristem-patterning gene, WUSCHEL. Stomata were frequently found within clusters of small cells and there was evidence for an extra cell division in some guard cells. Expression of PCNA, an S-phase gene, increased in RBR-downregulated leaves, but expression of the G2/M gene, CYCB2;2, was reduced. The proportion of nuclei with 2C DNA content decreased ∼2-fold suggesting that loss of RBR expression facilitated entry into S-phase. There was a corresponding increase in 4C nuclei, but higher ploidy levels were unchanged compared to empty vector controls. These results support a model in which cells continue to divide when RBR expression is reduced, even after acquiring characteristics of terminally differentiated cells, and suggest that RBR is needed for cell cycle exit.;In addition I have worked towards adapting our VIGS system to be used in a large-scale capacity consistent with the goals of functional genomics. To reach this goal we used recombinational cloning technologies to create VIGS vectors to 85 different Arabidopsis genes. VIGS constructs were systematically inoculated into Arabidopsis plants and phenotypic changes were observed. Because this was an exploratory survey, gene silencing was not confirmed by RT-PCR and the results must be verified before being accepted. A subset of the constructs created observable phenotypes, while many did not. Two examples of genes that showed silencing phenotypes are RIBOSOMAL PROTEIN S9 (RPS9), which showed chlorosis in new growth, and E2FB, which showed darkening at the base of trichomes in new leaves. Factors contributing to the lack of phenotypes for other genes include the limited time for observation, lack of quantitative data, potential for redundant gene function, interference with viral DNA replication or movement, and potential recalcitrance to gene silencing. Nevertheless, this type of screen could prove to be particularly useful in non-model systems that have limited genetic resources and for suppressor screens of genes such as RBR, which are gametophytic lethal. |
