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Fig. 6. Untagged dominant-negative mutant causes secretion of vacuole-targeted aleu-GFP, which can be rescued by AtRabF2b[wt]. Confocal images of tobacco leaf epidermal cells, 48-52 hours after Agrobacterium inoculation with aleu-GFP (OD₆₀₀ 0.1) and untagged-AtRabF2b and AtRabD2a (OD₆₀₀ 0.05). (A) With aleu-GFP alone, faint fluorescence is visible in some vacuoles (arrow). (B) In cells expressing aleu-GFP and AtRabF2b[wt], the fluorescent pattern of aleu-GFP is not affected. (C) With aleu-GFP and AtRabF2b[S24N], location of aleu-GFP is drastically altered by the dominant-negative mutant as fluorescence is clearly visible in the apoplast (arrow). (D) With aleu-GFP, AtRabF2b[S24N] and AtRabF2b[wt], AtRabF2b[wt] has rescued the effect of the dominant-negative mutant on the localization of aleu-GFP. Very few cells secreting GFP to the apoplastic space are present. (E) Cells expressing aleu-GFP, AtRabF2b[S24N] and AtRabD2a; expression of another Rab protein does not rescue the phenotype of aleu-GFP induced by AtRabF2b[S24N] as seen with AtRabF2b[wt]. Bar, 50 µm. (F) Analysis of cells with GFP visible in the apoplast. Data represent mean±s.d. of three independent experiments (eight fields of view per construct) of cells secreting to the apoplast. AL, aleu-GFP; WT, untagged AtRabF2b[wt]; SN, untagged AtRabF2b[S24N]; D2aWT, untagged AtRabD2a[wt].

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