Vector-based RNAi of RSP16. (A) The design of the RNAi construct. The RSP16 genomic DNA and cDNA fragments spanning exons 6 and 7 were ligated in an opposite orientation (black arrows) at each end of a spacer DNA that was PCR amplified from the A. thaliana EARLI1 gene. The hairpin fragment was cloned into the EcoRI site of the pSI103 vector upstream of the AphVIII expression cassette. The promotor activity in the opposite strand of the Rubisco 3′ untranslated region (T) should drive the expression (double-headed arrow) of the inverted sequence. Gray arrows and lines are reverse transcription primers and RT-PCR products for diagnosis of the transcript by RT-PCR. Dashed lines indicate the control, which detects amplification of genomic DNA. (B) Western blot screening RSP16 in the cell body extract from transformants displaying flagellar phenotypes. N, no flagella; P, paralyzed flagella; T, twitching flagella; and S, swimmers (control). The 50-kD cell body protein (arrowhead), which is also recognized by anti-RSP16 serum, served as a loading indicator and RSP16 in axonemes (arrow) was the positive control. Note that the 40-kD RSP16 was not discernable in the four twitching clones. (C) Western blots showed that the RSP16 band easily detected in the 20- and 5-μg wild-type axonemes was not detectable in ∼20-μg axonemes from the twitching clones. (bottom) Ponceau stain. (D) Transcription of hairpin construct was detected by RT-PCR of the exogenous loop using total RNA prepared from12E8 and the sense reverse transcription primer that could anneal to the predicted transcript. Control was using antisense primer (AS) that only annealed to genomic DNA.