RNA-Interference of Chlamydomonas reinhardtii RSP8

Matthew Greseth
Carroll College
Waukesha, WI
Mentor: Dr. Pinfen Yang

Motile cilia and flagella confer organisms superiority to mate, avoid predators, harvest food and respond to environmental stimuli.  The structure responsible for the motility within these organelles is the highly conserved 9+2 axoneme, which consists of nine outer doublet microtubules surrounding two central singlet microtubules.  Dynein arms anchored to the outer microtubules drive the microtubule sliding and consequently oscillatory beating.  However, the molecular mechanism for regulating dynein activation and thus flagellar beating remains largely unknown.

Studies of Chlamydomonas reinhardtii, a biflagellate unicellular green algae, suggested that the radial spoke, a T-shaped molecular complex regulates motility mechanically by interacting with the central pair and outer doublets as well as chemically by binding second messengers and changing enzyme activity.  A mutant, pf25, is defective in the gene encoding radial spoke protein (RSP) 11 that contains an RIIa domain known for anchoring cAMP-dependent protein kinase A to the kinase anchoring protein (AKAP).  RSP11 binds the spoke AKAP.  pf25 mutants displayed wild type motility in fresh medium, but became paralyzed in exhausted medium.  In contrast, wild type cells swim regardless.  In addition to RSP11, RSP8, an armadillo repeat spoke protein, is dramatically reduced as well.  To test that RSP11 regulates flagellar beating via RSP8, we use RNAi to knockdown RSP8.  Previously, the conventional hairpin plasmids failed possibly due to gene methylation.  New hairpin constructs and a paromomycin resistance cassette were inserted into the pHANNIBAL vector that was routinely used in RNAi of Arabidopsis. The single vector was transformed into Chlamydomonas.  Western blots of flagellar minipreps reveals antibiotic-resistant clones with RSP8 reduced at least 60%, indicating the pHannibal vector is suitable for RNAi of Chlamydomonas.  We are currently investigating the motility phenotype. 
 
 
 

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