DIR1 ER signal sequence directs secretion of the protein to the apoplast. Fusion proteins consisting of full length DIR1 fused to EYFP (DIR1-EYFP) and DIR1 lacking its signal sequence (DIR1Δ1-25 -EYFP) were expressed in Nicotiana tabaccum leaves via Agrobacterium-mediated transient expression. Fluorescent proteins were visualized in epidermal cells after 48 hours using confocal microscopy. DIR1-EYFP expression exhibited two distinct patterns. (A) Fluorescence in the region of the cortical ER and (D) the nuclear envelope and cell periphery. Expression of DIR1Δ1-25-EYFP and EYFP is shown in (B) and (C), respectively. Propidium iodide staining of the plant cell wall is illustrated in (E), and extensive colocalization of DIR1-EYFP with the propidium iodide signal is demonstrated in (F). Subcellular localization experiments were performed three times with similar results. (G) IWFs were collected from untreated leaves of 35S:DIR1Δ1-25-GUS-5/dir1-1 and DIR1pro:DIR1-GUS-29/dir1-1. GUS activity was determined by measuring the absorbance at 683 nm. This experiment was repeated 2 additional times with similar results.