Figure 4.

Inhibition of clathrin-mediated endocytosis in N. benthamiana seedlings by mRFP-Hub1 production opposes cell death. Seven-day-old seedlings were agroinfiltrated to produce mRFP-Hub1 (C,D), NtPhyt-EGFP (E,F), both proteins together (G,H), or neither of them (empty vector, A and B), and NtPhyt-S537A-EGFP (I,J). Three days after agroinfiltration, the seedlings were treated with 10 μM antimycin A for 3 h (the lower row) or mock-treated (buffer only control, the upper row). Subsequent staining with Evans Blue revealed marked cell death in the cotyledons upon antimycin A treatment (B,F,J), which was prevented by the mRFP-Hub1 production (D,H). Overproduction of NtPhyt-EGFP caused moderate damage by itself (E) and again, co-expression of mRFP-Hub1 nullified this effect (G). Overproduction of proteolytically inactive NtPhyt-S537A-EGFP mutant failed to cause cell death in the absence of antimycin A treatment (I). Data were reproducible over three independent experiments.