Plant Physiology 133:470-474 (2003)
© 2003 American Society of Plant Biologists
BREAKTHROUGH TECHNOLOGIES
RNA Interference in the Moss Physcomitrella patens1
Magdalena Bezanilla*,
Aihong Pan and
Ralph S. Quatrano
Department of Biology, Washington University, 1 Brookings Drive, Saint Louis, Missouri 63130-4899
The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP): -glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.
www.plantphysiol.org/cgi/doi/10.1104/pp.103.024901.
1 This work was supported by a post-doctoral research fellowship from the Helen Hay Whitney Foundation to M.B. and by the National Science Foundation (grant nos. IBN 0112461 to R.S.Q. and DBI 0216150 to Howard Berg from the Donald Danforth Plant Science Center Integrated Microscopy Facility, St. Louis).
* Corresponding author; e-mail manena{at}biology.wustl.edu; fax 314-935-5125.
Received April 4, 2003;
returned for revision May 6, 2003;
accepted July 8, 2003.
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