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. 2013 May 16;9(5):e1003378. doi: 10.1371/journal.ppat.1003378

Figure 3. Syp71 and Vap27-1 are recruited to the 6K2-induced elongated tubular structure.

Figure 3

(A) Localization of Syp71-YFP, Vap27-1-YFP and Syp72-YFP. Syp71-YFP (frame I), Vap27-1-YFP (frame II) and Syp72-YFP (frame III) were transiently expressed alone in N. benthamiana leaves. (B) 6K2-CFP and Syp72-YFP were transiently coexpressed in N. benthamiana leaves. (C and D) Syp71-YFP (yellow signal) was recruited to the elongated tubular structure containing 6K2-CFP (blue signal) at the junction between clumped chloroplasts (red autofluorescence) when they were coexpressed in N. benthamiana. (E) Syp71-mRFP (red signal) was recruited to the elongated tubular structure containing 6K2-GFP (green signal) at the junction between adjacent chloroplasts (autofluorescence signal was color-coded in blue) in N. benthamiana leaves infected by TuMV 6K2::GFP. (F and G) Vap27-1-YFP (yellow signal) was recruited to the elongated tubular structure containing 6K2-CFP (blue signal) at the junction between clumped chloroplasts (red autofluorescence) when 6K2-CFP and YFP-Syp71 were coexpressed in N. benthamiana. (H) Vap27-1-mRFP (red signal) was recruited to the elongated tubular structure containing 6K2-GFP (green signal) at the junction between adjacent chloroplasts (color-coded in blue) in N. benthamiana leaves infected by TuMV 6K2::GFP. Bars in (A through H), 8 µm. (I) Western blot analysis confirmed that Syp71-YFP was associated with chloroplasts when coexpressed with 6K2-CFP or during TuMV infection. The filters were detected by Syp71 antibodies (top panel), PsbA antibodies (middle panel) and Bip2 antibodies (bottom panel). 1, chloroplasts were purified from N. benthamiana leaves coexpressing Syp71-YFP and 6K2-CFP; 2, from leaves expressing Syp71-YFP and infected with TuMV::6K2-GFP; 3, from leaves expressing Syp71-YFP; P30, crude membrane fraction of leaves from mock-treated plants. PsbA and Bip2 serve as chloroplast and ER markers, respectively. (J) Western blot analysis confirmed that Vap27-1-YFP was associated with chloroplasts when coexpressed with 6K2-CFP or during TuMV infection. The filters were detected by Vap27-1 antibodies (top panel), PsbA antibodies (middle panel) and Bip2 antibodies (bottom panel). 1, chloroplasts were purified from N. benthamiana leaves coexpressing Vap27-1-YFP and 6K2-CFP; 2, from leaves expressing Vap27-1-YFP and infected with TuMV::6K2-GFP; 3, from leaves expressing Vap27-1-YFP; P30, crude membrane fraction.