in various eukaryotic cells, including yeast and plant cells [13, 15–
20]. Therefore, the tripartite split-sfGFP association presents a
highly specific readout of PPIs in vivo. Previous studies revealed
that the two engineered fragments of sfGFP, namely the GFP
β-strand 11 and the remaining complementary GFP1–10 fragment,
can self-assemble to achieve the split GFP protein tagging and
detection [9, 20]. Recently, we also discovered that the tripartite
split-sfGFP-based S11 and S10 tags exhibit the tendency to spontaneously associate the GFP1–10 fragments and the circularly permuted S11–GFP1–9 (Liu et al., unpublished data), respectively
(Fig. 1b). These findings provide an additional benefit of applying
the tripartite split-sfGFP tool for side-by-side analyses of in vivo
PPI detection along with in situ subcellular localization of fusion
proteins [18, 21]. Hence, constructing a full-length GFP protein
fusion and validating the protein expression level as well as its
localization are not a mandatory requirement prior to conducting
the tripartite split-sfGFP assay.
Compared to soluble PPIs, membrane PPIs is more technically
challenging to be uncovered. Because of the hydrophobic nature of
membrane proteins, biochemical and bioinformatics methods are
limited and/or close to saturation for identifying their genuine
interaction partners. Although the yeast mating-based split ubiquitin system (mbSUS) are the most widely used approach for plant
PPI analysis, a high rate of false positives and false negatives has
been reported [22–24]. In terms of minimizing unspecific behaviors, physiologically relevant interaction environment and proper
protein modification are preferred for the study of plant membrane
PPIs. Unlike the bulky fragments of the bipartite split-FP, the small
size of tripartite split-sfGFP-based S10 and S11 tags imposes a
minimal effect on membrane fusion protein behavior, thus conferring more precise information with regard to folding, solubility,
and targeting of membrane proteins [15]. Indeed, the feasibility of
tripartite split-sfGFP assay was recently validated for detection of
membrane PPIs at various cellular compartments in planta [18].
Although here we only describe protocols for the tripartite
split-sfGFP assay in leaf cells of Nicotiana benthamiana using the
Agrobacterium infiltration method, the application of tripartite
split-sfGFP system to transient transfection of plantprotoplasts is
also attainable.
2 Materials
2.1 Binary Vectors
and Constructs
1. PPI pairs of interest should be cloned into appropriate binary
vectors harboring β-Estradiol inducible tripartite split-sfGFP
fragments, which are deposited to Addgene (see Note 1). The
tripartite split-sfGFP-based binary vectors harboring a
3 Â Hemagglutinin (HA) tag are also available from Addgene
326
Tzu-Yin Liu
20]. Therefore, the tripartite split-sfGFP association presents a
highly specific readout of PPIs in vivo. Previous studies revealed
that the two engineered fragments of sfGFP, namely the GFP
β-strand 11 and the remaining complementary GFP1–10 fragment,
can self-assemble to achieve the split GFP protein tagging and
detection [9, 20]. Recently, we also discovered that the tripartite
split-sfGFP-based S11 and S10 tags exhibit the tendency to spontaneously associate the GFP1–10 fragments and the circularly permuted S11–GFP1–9 (Liu et al., unpublished data), respectively
(Fig. 1b). These findings provide an additional benefit of applying
the tripartite split-sfGFP tool for side-by-side analyses of in vivo
PPI detection along with in situ subcellular localization of fusion
proteins [18, 21]. Hence, constructing a full-length GFP protein
fusion and validating the protein expression level as well as its
localization are not a mandatory requirement prior to conducting
the tripartite split-sfGFP assay.
Compared to soluble PPIs, membrane PPIs is more technically
challenging to be uncovered. Because of the hydrophobic nature of
membrane proteins, biochemical and bioinformatics methods are
limited and/or close to saturation for identifying their genuine
interaction partners. Although the yeast mating-based split ubiquitin system (mbSUS) are the most widely used approach for plant
PPI analysis, a high rate of false positives and false negatives has
been reported [22–24]. In terms of minimizing unspecific behaviors, physiologically relevant interaction environment and proper
protein modification are preferred for the study of plant membrane
PPIs. Unlike the bulky fragments of the bipartite split-FP, the small
size of tripartite split-sfGFP-based S10 and S11 tags imposes a
minimal effect on membrane fusion protein behavior, thus conferring more precise information with regard to folding, solubility,
and targeting of membrane proteins [15]. Indeed, the feasibility of
tripartite split-sfGFP assay was recently validated for detection of
membrane PPIs at various cellular compartments in planta [18].
Although here we only describe protocols for the tripartite
split-sfGFP assay in leaf cells of Nicotiana benthamiana using the
Agrobacterium infiltration method, the application of tripartite
split-sfGFP system to transient transfection of plantprotoplasts is
also attainable.
2 Materials
2.1 Binary Vectors
and Constructs
1. PPI pairs of interest should be cloned into appropriate binary
vectors harboring β-Estradiol inducible tripartite split-sfGFP
fragments, which are deposited to Addgene (see Note 1). The
tripartite split-sfGFP-based binary vectors harboring a
3 Â Hemagglutinin (HA) tag are also available from Addgene
326
Tzu-Yin Liu