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. 2017 Feb 28;6:e23897. doi: 10.7554/eLife.23897

Figure 1. CLCuMuB βC1 interacts with NbATG8f in vivo and in vitro.

(A) βC1 interacts with NbATG8f in yeast. SKY48 yeast strains containing AD-NbATG8f transformed with BD-βC1 or BD (control) were grown on Leu- selection plates at 28°C for 4 d. The positive interaction was indicated by the blue colony formation on X-gal-containing galactose (Gala) and raffinose (Raf) but not on plates containing glucose (Glu). (B) GST pull-down assay to show the in vitro interaction of NbATG8f with βC1, but not βC1V32A. The total soluble proteins of E. coli expressing NbATG8f-6×His were incubated with GST-βC1 or GST-βC1V32A immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) βC1 was co-immunoprecipitated with NbATG8f. GFP-NbATG8f was transiently co-expressed with and HA-βC1 or its mutant HA-βC1V32A in N. benthamiana leaves. At 60 hr post agroinfiltration (hpi), leaf lysates were immunoprecipitated with anti-GFP beads and then the precipitants were assessed by immunoblotting (IB) using anti-HA (upper panel) or anti-GFP antibodies (middle panel). (D) BiFC analyses in N. benthamiana. Representative images of nYFP-βC1 or nYFP-βC1V32A BiFC co-expressed with cYFP-NbATG8f. (E) Western blot analyses of BiFC construct combinations from the same experiments as in (D). All combinations were detected with anti-GFP polyclonal antibody.

DOI: http://dx.doi.org/10.7554/eLife.23897.003

Figure 1.

Figure 1—figure supplement 1. N terminus of βC1 is responsible for binding to NbATG8f.

Figure 1—figure supplement 1.

Schematic representation of the truncated mutants of βC1, their interactions with NbATG8f in yeast. Yeast cells transformed with four truncated mutants of βC1 and NbATG8f were selected on X-Gal-containing medium.
DOI: http://dx.doi.org/10.7554/eLife.23897.004
Figure 1—figure supplement 2. βC1 co-immunoprecipitated with multiple ATG8 homologs.

Figure 1—figure supplement 2.

(A) Homology tree of NbATG8s. Homology tree of eight homologs of NbATG8 in N. benthamiana. The results were produced by DNAMAN package and the observed divergency method was applied to calculate distance. (B) GFP-NbATG8s were transiently co-expressed with HA-βC1 in N. benthamiana leaves. At 60 hpi, leaf lysates were immunoprecipitated with anti-GFP beads and then the precipitants were assessed by immunoblotting (IB) using anti-HA (upper panel) or anti-GFP antibodies (middle panel).
DOI: http://dx.doi.org/10.7554/eLife.23897.005
Figure 1—figure supplement 3. βC1 is co-localized with NbATG8f.

Figure 1—figure supplement 3.

CFP-NbATG8f was transiently co-expressed with YFP-βC1 or YFP-βC1V32A in N. benthamiana leaves via agroinfiltration. The confocal microscope images of mesophyll cells were taken at 60 hpi.
DOI: http://dx.doi.org/10.7554/eLife.23897.006