Fig. 5. The Arabidopsis HECs interact with SPT in yeast and are negatively regulated by ETT. (A-C) The HECs are still expressed (arrowheads) in the septum in the spt-2 mutant. HEC1 expression (A) was analyzed by crossing the HEC1p:HEC1:GUS line into spt-2, and HEC2 (B) and HEC3 (C) expression was analyzed directly by in situ hybridization. (D-F) The HECs are ectopically expressed in abaxial cell layers of the gynoecium (arrowheads) in ett-7. HEC1 expression (D) was analyzed by crossing the HEC1p:HEC1:GUS line into spt-2, and HEC2 (E) and HEC3 (F) expression was analyzed directly by in situ hybridization. Expression could also still be seen in the septum (arrows) for HEC1 (D) and HEC2 (E). (G) The HEC proteins do not homodimerize or heterodimerize with each other in a yeast two-hybrid system. Full-length HEC1 and HEC2 were used in both the bait and prey constructs. Full-length HEC3 was used as prey. However, an N-terminal deletion of HEC3 was used as the bait, as the full-length HEC3 bait construct activated the yeast reporter genes on its own (data not shown). The protein cruciferin was used as a negative control. Results were confirmed with the HIS3 reporter. (H) The HEC proteins form heterodimers with SPT in a yeast two-hybrid system. An N-terminal deletion of SPT was used as the prey construct (see Materials and methods). The protein cruciferin was used as a negative control. Results were confirmed with the HIS3 reporter. Scale bars: 50 µm.