within a radius of ~10 nm [3, 4]. Biotinylated proteins can be
extracted from cells using stringent extraction and solubilization
methods without the need to maintain protein-protein interactions, providing access to membrane-associated and insoluble protein complexes. Biotinylated proteins can then be affinity purified
using streptavidin beads and identified using mass spectrometry
and/or western blot.
The power of BioID as a proteome mapping tool has been
demonstrated for a number of biological systems [1, 2, 5, 6]. For
example, BioID baits have been used to characterize the composition of the nuclear lamina [2], to generating a global Zika virushost protein interactome [7], and also to identify the organization
and composition of P-bodies and stress granules [8]. BioID has also
been applied to a variety of organisms including mammalian cells,
Trypanosoma brucei, Toxoplasma gondii, Dictyostelium discoideum,
Theileria annulata, Plasmodium spp., and Saccharomyces cerevisiae
[5, 9–11]. In plants, BioID has been developed in three systems:
rice protoplasts [12], stable transgenic Arabidopsis thaliana tissue
[13], and transient expression in Nicotiana benthamiana tissues
[14]. Lin et al., 2017, used BioID in rice protoplasts with a modified BirA* called BirAG which removed a cryptic intron splicing
site. Using this modified BirAG, they characterized the proteome
network of OsFD2, a protein involved in vegetative growth in rice
[12]. Conlan et al., 2018, used BioID to identify potential immune
system components of N. benthamiana using the AvrPto
Pst effector
protein from Pseudomonas syringae as bait [14]. In Arabidopsis, we
used BioID to identify host protein targets of P. syringae effector
HopF2b
PtoDC3000 [13]. Although all three of these studies applied
BioID in planta, there are variations in the protocols used due to
differences in protein expression systems, plant species, as well as
plant materials used (Table 1). Below we describe the BioID protocol for A. thaliana tissues that was published in Khan et al., 2018
[13] (Fig. 1). This protocol was developed to identify proteins
interacting with the membrane-associated P. syringae effector protein HopF2b
PtoDC3000 from mature leaf tissues. The use of BioID
in planta has focused on using specific bait proteins to identify their
interacting proteins (Fig. 1). However, this technique can be
expanded to identify global proteomes of compartments and organelles in the plant cell (Fig. 1). It should be noted that recent
modifications to BioID were developed called TurboID and miniTurbo which were experimentally evolved from BirA to allow for
more efficient biotinylation and reduced labeling time from 18 h to
as low as 10 min [15]. Three recent manuscripts present the use of
TurboID to study the proteome proximal to N, a nucleotidebinding leucine-rich repeat (NLR) protein that confers resistance
to Tobacco Mosaic Virus, organellar proteome, and BIN2 kinase
signaling network in plants [16–18]. Although this chapter focuses
426
Madiha Khan et al.
extracted from cells using stringent extraction and solubilization
methods without the need to maintain protein-protein interactions, providing access to membrane-associated and insoluble protein complexes. Biotinylated proteins can then be affinity purified
using streptavidin beads and identified using mass spectrometry
and/or western blot.
The power of BioID as a proteome mapping tool has been
demonstrated for a number of biological systems [1, 2, 5, 6]. For
example, BioID baits have been used to characterize the composition of the nuclear lamina [2], to generating a global Zika virushost protein interactome [7], and also to identify the organization
and composition of P-bodies and stress granules [8]. BioID has also
been applied to a variety of organisms including mammalian cells,
Trypanosoma brucei, Toxoplasma gondii, Dictyostelium discoideum,
Theileria annulata, Plasmodium spp., and Saccharomyces cerevisiae
[5, 9–11]. In plants, BioID has been developed in three systems:
rice protoplasts [12], stable transgenic Arabidopsis thaliana tissue
[13], and transient expression in Nicotiana benthamiana tissues
[14]. Lin et al., 2017, used BioID in rice protoplasts with a modified BirA* called BirAG which removed a cryptic intron splicing
site. Using this modified BirAG, they characterized the proteome
network of OsFD2, a protein involved in vegetative growth in rice
[12]. Conlan et al., 2018, used BioID to identify potential immune
system components of N. benthamiana using the AvrPto
Pst effector
protein from Pseudomonas syringae as bait [14]. In Arabidopsis, we
used BioID to identify host protein targets of P. syringae effector
HopF2b
PtoDC3000 [13]. Although all three of these studies applied
BioID in planta, there are variations in the protocols used due to
differences in protein expression systems, plant species, as well as
plant materials used (Table 1). Below we describe the BioID protocol for A. thaliana tissues that was published in Khan et al., 2018
[13] (Fig. 1). This protocol was developed to identify proteins
interacting with the membrane-associated P. syringae effector protein HopF2b
PtoDC3000 from mature leaf tissues. The use of BioID
in planta has focused on using specific bait proteins to identify their
interacting proteins (Fig. 1). However, this technique can be
expanded to identify global proteomes of compartments and organelles in the plant cell (Fig. 1). It should be noted that recent
modifications to BioID were developed called TurboID and miniTurbo which were experimentally evolved from BirA to allow for
more efficient biotinylation and reduced labeling time from 18 h to
as low as 10 min [15]. Three recent manuscripts present the use of
TurboID to study the proteome proximal to N, a nucleotidebinding leucine-rich repeat (NLR) protein that confers resistance
to Tobacco Mosaic Virus, organellar proteome, and BIN2 kinase
signaling network in plants [16–18]. Although this chapter focuses
426
Madiha Khan et al.