Geminivirus-induced Gene Silencing as a Method to Determine the Role of Essential Cell Cycle Genes in Plant Development

Abstract

Tomato Golden Mosaic Virus (TGMV) is a DNA virus that replicates and transcribes its genome in plant nuclei. TGMV was modified to serve as a gene silencing vector in Nicotiana benthamiana by insertion of small fragments (120 — 160 bp) downstream of the viral BR1 gene. Inoculation by microprojectile bombardment resulted in local silencing after 5-7 days and persistent systemic silencing. Previously, TGMV vectors were used to silence genes needed for chlorophyll biosynthesis. The purpose of my research was to determine if TGMV vectors could be used to provide information about genes essential for DNA replication and plant development.

I tested TGMV vectors containing 120 — 150 bp inserts from genes encoding the N. benthamiana proliferating cell nuclear antigen (PCNA) and N. tabacum retinoblastoma-related protein (pRBR). PCNA is essential for DNA replication and pRBR is needed for cell cycle control. TGMV-mediated silencing of PCNA resulted in an irreversible cessation of primary growth. Leaves were clustered, showed truncated expansion, and fused with the stem at their base. Meristematic-like structures that were elongate, lacked leaf primordia, and contained files of cells that did not expand were found, but only in a small proportion of silenced plants. Immunolocalization demonstrated that PCNA expression was silenced throughout these structures. These experiments provided the first evidence that TGMVinduced silencing could down-regulate expression in meristems, and suggested that proper PCNA expression could be required for cell expansion as well as primary growth.

TGMV-mediated gene silencing of RBR resulted in delayed programmed cell death in mature tissues, as demonstrated by Trypan blue staining. Wild type TGMV infectionsproduced severe symptoms but did not result in cell death. Systemically silenced leaves of TGMV::RBR inoculated plants had developmental defects that were distinct from symptoms. Flower corollas were abnormally curled and some flowers were infertile. A low but reproducible proportion of progeny derived from the seed of curled flowers also had abnormal leaf growth suggestive of an epigenetic modification. The onset of cell death in mature tissues of pRBR-silenced plants and the developmental defects in new growth suggests that pRBR may have distinct roles in dividing cells and differentiated tissues.