Poster: Emerging Technologies

Abs # 889: RNAi in the Moss Physcomitrella patens

Presenter:	Bezanilla, Magdalena , manena@biology.wustl.edu
Authors	Bezanilla, Magdalena  (A)   Quatrano, Ralph S.  (A)
Affiliations:	(A): Washington University, Department of Biology

Physcomitrella patens performs efficient homologous recombination, allowing for targeted gene disruption and functional analysis of individual genes. However, as in higher plants, gene families in Physcomitrella can be quite large. RNAi has been used successfully in higher plants to disrupt the function of large gene families. We show here that RNAi disrupts gene expression in moss. We constructed a line (NLS-4) stably expressing a nuclearly localized GUS-GFP fusion reporter protein. NLS-4 has green fluorescence in the nucleus at all stages of haploid growth and has no visible phenotype compared to wild type. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. These constructs are designed to produce double stranded RNA hairpins driven by the maize ubiquitin promoter. Transformed cells are marked by co-bombardment with dsRed, which freely diffuses between the nucleus and cytoplasm. In control experiments transformed cells 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 ~ 10-fold as soon as 24 hours after transformation. When co-bombarding with luciferase-RNAi control construct, the nuclear green fluorescence is quantitatively not affected. This result demonstrates that Physcomitrella carries out RNAi as in higher plants. We are currently testing whether stably integrated RNAi constructs can down regulate gene expression in Physcomitrella. Stable RNAi expression will allow for functional analysis of large gene families by observing phenotypic consequences of reduced gene expression.