Silencing of a meristematic gene using geminivirus-derived vectors

Geminiviruses 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 silen cing from a bipartite geminivirus in Nicotiana benthamiana. Using an improv ed 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 la rger inserts producing milder symptoms. In situ hybridization of silenced t issue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral in fection. We also show that two essential genes encoding a subunit of magnes ium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunol ocalization of silenced tissue showed that the PCNA protein was down-regula ted throughout meristematic tissues. Our results demonstrate that geminivir us-derived vectors can be used to study genes involved in meristem function in intact plants.