Widefield microscopy showed Jonah(1)-GFP and glycopolymers labeled by MBP-Jonah(1) were accessible in the ectocyst layer of mature cyst walls, while Luke(2)-GFP, Leo-GFP, and glycopolymers labeled by MBP-Luke(2) and MBP-Leo were inaccessible in the endocyst layer and ostioles.

A. Nearly 100% of mature cysts expressing Jonah(1)-GFP (green) under its own promoter were labeled with anti-GFP antibodies (red). CFW (blue) labeled the endocyst layer of cyst walls. B. Just 3% of mature cysts expressing Luke(2)-GFP under its own promoter labeled with anti-GFP antibodies. C. Just 2% of mature cysts expressing Leo-GFP under its own promoter labeled with anti-GFP antibodies. D. MBP-Jonah(1) (red) labeled 100% of mature cysts, which were also labeled with WGA (green) and CFW. In contrast, MBP-Luke(2) (E) and MBP-Leo (F) each labeled 9% of mature cysts. While it was sometimes possible to distinguish the ectocyst and endocyst layers with widefield and DIC microscopy, it was not easy. Similarly, it was very difficult to identify ostioles with widefield microscopy and impossible with DIC. A to F. Scale bars are 5 μm. SIM of mature cysts labeled with MBP-lectin fusions are shown in S7 Fig.