inserted into a variety of coding sequences to regulate gene expression in plants. Further analysis has revealed that cassette function apparently requires conservation of only two upstream nucleotides in the coding sequence, making this conditional splicing system quite general. Finally, we demonstrate that strong gene activation ( 97-fold) can be accomplished by alternative splicing using an optimized version of the suicide exon. MATERIALS AND METHODS Oligonucleotides and DNA constructs Sequences of all synthetic primers used for making and mutagenizing DNA constructs, performing overlap exten- sion PCR, RT–PCR and RT–qPCR are described in Supplementary Table S1. All constructs conﬁrmed by sequencing are shown in Supplementary Data. TFIIIA-eGFP (formerly called Pre-EGFP), LUC (used as a control except when assaying the fLUC reporter), AtL5 and DsRed2 constructs were described previously (23). The coding sequence of OsL5 (Os01g0896800) also was previously isolated and cloned into the pBinAR vector (23). The fLUC coding sequence was derived from the pGL3 vector (Promega). A. thaliana coding se- quences were ampliﬁed by PCR from cDNA (see RNA isolation methods) and cloned using the TOPO TA Cloning kit (Invitrogen). Mutations to TFIIIA-eGFP were made using the QuikChange II Site-Directed Mutagenesis (Stratagene) or QuikChange Lightning Site-Directed Mutagenesis kits (Stratagene) according to the manufacturer’s instructions. The P5SM splicing cassette sequence was inserted into constructs by overlap extension PCR (24). Brieﬂy, the coding sequence to be modiﬁed was ampliﬁed as two DNA fragments using PCR primers that introduce a region that overlaps with the sequence of the P5SM-splicing cassette. For the front fragment, the overlapping region is at the 30-end, and for the back fragment, the overlapping region is at the 50-end. The P5SM-splicing cassette was ampliﬁed by PCR using TFIIIA-eGFP as the DNA template, generating a third DNA fragment. The DNA fragments were puriﬁed by agarose gel electrophoresis and then mixed in a 1:1:1 ratio as partial templates for overlap extension PCR, which uses forward and reverse primers that anneal to the 50- and 30-ends of the desired full-length construct. The PCR product containing the P5SM cassette was puriﬁed by agarose gel electrophoresis before being cloned into the TOPO vector for sequence conﬁrmation. To generate constructs for use in plant inﬁltration experi- ments, they were re-ampliﬁed to introduce restriction sites at the ends (Supplementary Table S1), digested with the appropriate restriction enzymes, and ligated using T4 DNA ligase (NEB) into the binary vector pBinAR (25). The sequence encoding the P5SM RNA element from O. sativa was derived from the genomic fragment of Os02g0116000 previously cloned into the TOPO vector (23). To generate the eGFP-OsP5SM construct, overlap extension PCR was performed to insert OsP5SM in place of the original P5SM from A. thaliana. To Nucleic Acids Research, 2012, Vol. 40, No. 10 4703 generate eGFP-HyP5SM, the purine-rich insertion was introduced directly into the primers used to amplify eGFP-OsP5SM as two DNA fragments, then the two overlapping pieces were extended by PCR. After sequence conﬁrmation of these constructs in the TOPO vector, they were cloned into pBinAR as described above. Leaf-based ﬂuorescence assay In vivo reporter ﬂuorescence assays were performed by Agrobacterium-mediated leaf inﬁltration in Nicotiana benthamiana as previously described (19). Brieﬂy, each leaf half was inﬁltrated with a 1:1:1 mixture of Agrobacterium transformed with pBinAR plasmids carrying the reporter construct, inducer (AtL5 or OsL5) or the control (LUC), and DsRed2 as a normalization standard. For assaying the fLUC reporter, untransformed Agrobacterium was used as the control instead. In vivo ﬂuorescence was measured 3 days post-inﬁltration using the Typhoon laser-based scanning system (GE Healthcare) using excitation and emission wavelength settings of 488/520 nm for eGFP and 532/580 nm for DsRed2. The ratios of eGFP/DsRed2 ﬂuorescence readings were taken for each leaf half in order to normal- ize differences in transformation efﬁciency. On average, 15% difference was observed between DsRed2 ﬂuores- cence for leaf halves from the same leaf sample. To calculate relative fold induction by AtL5 or OsL5, the eGFP/DsRed2 ratio for the leaf half coexpressing the inducer was divided by the other half coexpressing the con- trol. The average relative fold induction was calculated from these values for several leaf samples, and the number of independent leaf samples analyzed (n) and standard deviations are as indicated. No more than two leaf samples were analyzed from the same plant. Autoﬂuorescence (deﬁned as ﬂuorescence measured for uninﬁltrated, or ‘blank’, leaves) was not measured, and so was not subtracted from the eGFP ﬂuorescence readings for all experiments except for the one showing relative ﬂuorescence of HyP5SM without DsRed2. For this experiment, autoﬂuorescence was measured and sub- tracted from the eGFP ﬂuorescence readings of both leaf halves, and fold induction for each individual leaf was calculated by taking the ratio of the half coexpressing OsL5 versus the other coexpressing LUC (control). RNA isolation and RT–PCR analyses Total RNA was isolated from 100 mg N. benthamiana leaf tissue collected 2 days post-inﬁltration using the Universal RNA Puriﬁcation Kit (CHIMERX) according to the manufacturer’s instructions. Leaf tissue samples were immediately frozen in liquid nitrogen after being placed in microtubes, then pulverized using a bead mill (TissueLyser, Qiagen) prior to RNA extraction. The samples were kept frozen during the milling process by being placed in an adapter rack that was pre-chilled at 80 C. The entire procedure was performed as quickly as possible, and the integrity of the RNA after isolation was checked by analysis on an agarose gel. cDNA was generated from 500 ng RNA samples that had been treated with RQ1 DNase (Promega) using