Two positive clones were sequenced and found to include 219 amino acids that are identical to the C terminus of At3g44110, which encodes a putative cochaperone DnaJ like heat shock protein . To narrow down the interaction domain in J3, J3C 219 was divided into two parts, J3C1 and J3C2 ; the structures of the peptides are shown in Figure 1A. These fragments and the full length J3 were cloned into the pACT2 vector, and combinations of PKS5 and J3 were cotransformed into yeast. Both the J3C1 and J3C2 peptides interacted with the full length PKS5 protein and the C terminus of PKS5, with J3C1 showing a stronger interaction than J3C2 . The PKS5 protein interacted weakly with J3 . To determine the region of PKS5 that interacts with J3, fragments encoding the N terminal kinase domain or the C terminal regulatory domain of PKS5 were cloned into pAS2, and these two plasmids were cotransformed with the J3 plasmids into yeast. The PKS5 kinase domain did not interact with any portion of J3 . The PKS5 C terminus interacted with J3C1, which showed a stronger interaction than any other J3 fragment .
As controls, PKS5 was shown to masitinib structure selleck chemicals interact with SOS3 LIKE CALCIUM BINDING PROTEIN1 , and this interaction was abolished when the FISL domain was deleted . To determine if this interaction exists in vivo, three FLAG tags in a tandem repeat were fused to PKS5 or to the trichomeassociated gene TRANSPARENT TESTA GLABRA1 at their Ntermini, and six Myc tags in a tandem repeat were fused to J3 at its N terminus with fusions for all three genes under the control of the 35S promoter. Combinations of 63Myc J3 and 33FLAG TTG1 or 63Myc J3 and 33FLAG PKS5 were cotransfected into Arabidopsis leaf protoplasts. The 63Myc J3 protein was immunoprecipitated using anti Myc conjugated agarose. After washing, immunoblots were probed with anti FLAG antibody. The 33FLAG PKS5 but not 33FLAG TTG1 protein was pulled down by 63Myc J3 , suggesting that PKS5 and J3 can function in the same complex. Together with the yeast two hybrid results, our data indicate that PKS5 and J3 interact in vivo.
PKS5 and J3 Have Overlapping Tissue Specific Expression and Subcellular Localization To determine if PKS5 and J3 colocalize in planta, we monitored PKS5 and J3 tissue specific expression using two approaches. First, a 1918 bp DNA fragment dyphylline upstream of the J3 translational start codon was amplified and cloned into pCambia1301 transcriptionally fused to b glucuronidase and the resulting plasmid was transformed into Columbia 0 . GUS signals driven by the PKS5 or J3 promoter are shown in Figures 2A to 2E and 2F to 2J, respectively. Both J3P: GUS and PKS5P:GUS were expressed in the roots and leaves of seedlings with stronger GUSstaining in vascular tissue .