Poster: Tropisms

Abs # 677: RNAi analysis of polarity development in Ceratopteris richardii

Presenter:	Stout, Stephen C, stephenstout@mail.utexas.edu
Authors	Stout, Stephen C (A)   Clark, Greg B (A)   Porterfield, Marshall  (B)   Roux, Stanley J (A)
Affiliations:	(A): University of Texas (B): University of Missouri, Rolla

We are developing RNAi, or RNA silencing, as a technique to investigate gene function during gravity directed polarity development in the single celled spores of Ceratopteris richardii. We incubated germinating spores with various concentrations of double stranded RNA constructs derived from the sequences of three genes from Ceratopteris: a calmodulin, a calmodulin-domain protein kinase, and a profilin. dsRNA concentrations of 0.05 mg mL^-1 to 0.10 mg mL^-1 specifically suppress the expression of the target gene at the mRNA level as measured by RT-PCR. Suppression occurs within 1 to 2 h and can last up to 120 h, indicating that this technique may be useful for studying the events of gravity perception and response, which occur during a limited period between 6 and 24 h after initiation of germination. Pulse-chase experiments indicate that the spores are permeable to dsRNA at least within the first 36 h of development and that gene silencing requires exogenous dsRNA. Subsequent experiments will examine changes in gene expression at the protein level, as well as alternative strategies for inducing RNA silencing in Ceratopteris. However, these results indicate that RNA silencing is a promising approach for investigating the molecular basis of gravity perception and signal transduction in Ceratopteris richardii.