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THE USE OF RNAi TO INVESTIGATE GENE FUNCTION DURING POLARITY DEVELOPMENT IN CERATOPTERIS RICHARDII.  S.C. Stout and S.J. Roux.  Molecular Cell and Developmental Biology, University of Texas at Austin.

   We are developing RNA silencing as a technique to investigate gene function during gravity directed polarity development in the single celled spores of Ceratopteris richardii.  In order to determine if RNA silencing, a post-transcriptional gene silencing mechanism, may be used in this system, we examined whether or not protein translation was required for spore germination.  ³⁵S labeled methionine incorporation revealed there was significant protein synthesis during the first 24 h of germination, and transient cycloheximide treatments delayed or prevented spore germination.  These results indicate that new protein synthesis is needed for germination progression, and thus, that RNA silencing may be used to examine the role of specific genes during germination in Ceratopteris.  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 0.5 mg mL^-1 to 1.0 mg mL^-1 specifically suppress the expression of the target gene at the mRNA level.  Additional experiments are necessary to determine the duration of suppression and the amount of suppression observed at the protein level.  Nonetheless, these results indicate that RNA silencing is a promising approach for investigating the molecular basis of gravity perception and signal transduction in Ceratopteris richardii.

(Supported by NAG10-0295, NAG2-1347 to SJR and NGT5-50371 to SCS.)