Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level.
Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.
Qi
Yijun
Y
Department of Plant Cellular and Molecular Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, USA. qiy@cshl.edu
Zhong
Xuehua
X
Itaya
Asuka
A
Ding
Biao
B
D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D011523:Protoplasts / Q000378:metabolism / Q000821:virology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000821:virology; D014764:Viral Proteins / Q000378:metabolism*
D034741:RNA, Small Interfering; D014764:Viral Proteins