Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana

• Published in: Journal of experimental botany Vol. 65; no. 15; pp. 4271 - 4283
• Main Authors: Sohn, Seong-Han, Frost, Jennifer, Kim, Yoon-Hee, Choi, Seung-Kook, Lee, Yi, Seo, Mi-Suk, Lim, Sun-Hyung, Choi, Yeonhee, Kim, Kook-Hyung, Lomonossoff, George
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.2014
• Subjects: Gene expression; Gene Expression Profiling; Gene Silencing; Genes; Green Fluorescent Proteins; Nicotiana - genetics; Phenotype; Phenotypes; Plants, Genetically Modified; Proteins; Repetitive Sequences, Nucleic Acid; RESEARCH PAPER; RNA; RNA interference; Small interfering RNA; Transgenes; Transgenic plants; Vascular plants; cell-autonomous-like silencing; Nicotiana benthamiana; GFP-partitioned silencing; systemic silencing; post-transcriptional gene silencing
• ISSN: 0022-0957; 1460-2431
• DOI: 10.1093/jxb/eru200

We previously reported the novel partitioning of regional GFP-silencing on leaves of 35S–GFP transgenic plants, coining the term "partitioned silencing". We set out to delineate the mechanism of partitioned silencing. Here, we report that the partitioned plants were hemizygous for the transgene, possessing two direct-repeat copies of 35S–GFP. The detection of both siRNA expression (21 and 24 nt) and DNA methylation enrichment specifically at silenced regions indicated that both post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) were involved in the silencing mechanism. Using in vivo agroinfiltration of 35S–GFP/GUS and inoculation of TMV–GFP RNA, we demonstrate that PTGS, not TGS, plays a dominant role in the partitioned silencing, concluding that the underlying mechanism of partitioned silencing is analogous to RNA-directed DNA methylation (RdDM). The initial pattern of partitioned silencing was tightly maintained in a cell-autonomous manner, although partitioned-silenced regions possess a potential for systemic spread. Surprisingly, transcriptome profiling through next-generation sequencing demonstrated that expression levels of most genes involved in the silencing pathway were similar in both GFP-expressing and silenced regions although a diverse set of region-specific transcripts were detected.This suggests that partitioned silencing can be triggered and regulated by genes other than the genes involved in the silencing pathway.