The regulation of PCNA expression during plant development and geminivirus infection

Many aspects of cell cycle control are conserved between animals and plants. Components of the pRb-E2F pathway have been found in a number of plant species. Like the mammalian DNA tumor viruses, the plant geminivirus tomato golden mosaic virus (TGMV) must recreate a replication competent environment to amplify its genome. TGMV does this in part by disrupting the pRb/E2F pathway. TGMV must make extensive use of the host replication machinery for its replication because of its limited coding capacity. TGMV induces accumulation of the DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) in mature plant tissues that do not normally produce it. The purpose of this project was to examine the mechanism of PCNA induction by TGMV. The PCNA gene from Nicotiana benthamiana was isolated and its expression was examined. TGMV induction was at the level of transcription, with viral infection resulting in a secondary peak of PCNA expression in mature leaves, which correlates with symptom severity and viral DNA levels. The PCNA promoter contains two E2F consensus binding sites that bind to proteins in N. benthamiana nuclear extracts. The roles and interaction of two E2F sites were examined in transgenic plants. In mature leaves, mutation of either site or both sites simultaneously resulted in relief of repression. In young tissues, mutation of the distal E2F site decreased expression, while mutation of the proximal site or both sites together had no effect. Mutation of the E2F sites prevented any detectable effect of TGMV infection. These results demonstrated that both E2F elements contribute to repression of the PCNA promoter in mature leaves whereas the distal E2F site acts as an antirepressor in young leaves to counter the repression activity of the proximal element. The role of E2F in regulation at all stages of plant development is supported by immunohistochemical staining that located both E2F and DP, a protein that associates with E2F to promote efficient DNA binding, in young and mature plant tissues.