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Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors.

Gookin TE, Assmann SM - Plant J. (2014)

Bottom Line: Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives.We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells.We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 'DRY' motif abolishes both of these interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Pennsylvania State University, University Park, PA, 16802, USA.

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Related in: MedlinePlus

GPA1 interacts with PLDα1, but not with the PLDm1 (EKF→GAA) residue substitution mutant.(a) The GPA1:NmVen210—X:CVen210 parent vector shows no background BiFC signal even when agroinfiltrated into Nicotiana benthamiana leaves at an optical density of 0.2 and imaged 66 h post-infiltration; maximum projection shows blue XT–Golgi–mTq2 signal marking transformed cells.(b) GPA1:NmVen210 interacts with PLDα1:CVen210; maximum projection shows the signal is only at the cell periphery.(c) GPA1:NmVen210 appears not to interact with PLDm1:CVen210, but the presence of transformed cells cannot be confirmed when p19 is in MCS2.(d) GPA1:NmVen210 does not interact with PLDm1:CVen210; the mTq2 Golgi marker clearly identifies transformed cells, confirming a true negative event even when few cells are transformed.(e) GPA1:NmVen210 interaction with PLDa1::CVen210 occurs in Arabidopsis gpa1-3 mesophyll protoplasts.(f) GPA1:NmVen210 does not interact with PLDm1:CVen210 in Arabidopsis gpa1-3 mesophyll protoplasts.(g) GPA1:NmVen210 also does not interact with PLDm2 in Arabidopsis gpa1-3 mesophyll protoplasts. Agroinfiltration of N. benthamiana at very low optical densities (0.005–0.01) allows interaction assays (b, d) in well-isolated cells 44 h post-infiltration. Arabidopsis protoplasts were imaged 12 h post-transformation. Maximum projections (a) ×20 and (b) ×40 magnifications with digital zoom. Single focal plane images at (c, d) ×40 and (e–g) at ×63 magnifications. Identical mVenus channel parameters for (e–g) allow direct comparison. All scale bars = 20 μm except (c) = 50 μm. Yellow = mVenus BiFC, blue = mTq2 Golgi marker, red = autofluorescence.
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fig06: GPA1 interacts with PLDα1, but not with the PLDm1 (EKF→GAA) residue substitution mutant.(a) The GPA1:NmVen210—X:CVen210 parent vector shows no background BiFC signal even when agroinfiltrated into Nicotiana benthamiana leaves at an optical density of 0.2 and imaged 66 h post-infiltration; maximum projection shows blue XT–Golgi–mTq2 signal marking transformed cells.(b) GPA1:NmVen210 interacts with PLDα1:CVen210; maximum projection shows the signal is only at the cell periphery.(c) GPA1:NmVen210 appears not to interact with PLDm1:CVen210, but the presence of transformed cells cannot be confirmed when p19 is in MCS2.(d) GPA1:NmVen210 does not interact with PLDm1:CVen210; the mTq2 Golgi marker clearly identifies transformed cells, confirming a true negative event even when few cells are transformed.(e) GPA1:NmVen210 interaction with PLDa1::CVen210 occurs in Arabidopsis gpa1-3 mesophyll protoplasts.(f) GPA1:NmVen210 does not interact with PLDm1:CVen210 in Arabidopsis gpa1-3 mesophyll protoplasts.(g) GPA1:NmVen210 also does not interact with PLDm2 in Arabidopsis gpa1-3 mesophyll protoplasts. Agroinfiltration of N. benthamiana at very low optical densities (0.005–0.01) allows interaction assays (b, d) in well-isolated cells 44 h post-infiltration. Arabidopsis protoplasts were imaged 12 h post-transformation. Maximum projections (a) ×20 and (b) ×40 magnifications with digital zoom. Single focal plane images at (c, d) ×40 and (e–g) at ×63 magnifications. Identical mVenus channel parameters for (e–g) allow direct comparison. All scale bars = 20 μm except (c) = 50 μm. Yellow = mVenus BiFC, blue = mTq2 Golgi marker, red = autofluorescence.

Mentions: We cloned Arabidopsis wild-type PLDα1 into MCS3 of our GPA1 parent vector to produce a BiFC construct in the GPA1:NmVen210—PLDα1:CVen210 configuration. Using site-directed mutagenesis, we also created PLDα1 mutant versions containing an EKF→GAA substitution (PLDm1) and a longer EKFRVY→GAASGS substitution (PLDm2) which also destroys part of an adjacent highly conserved hydrophobic patch. Importantly, these single vector constructs all contain an integrated Golgi-localized mTurquoise2 marker (XT-Golgi-mTq2) to specifically identify transformed cells. Agroinfiltration into N. benthamiana leaves showed that GPA1 can interact with wild-type PLDα1 in living plant cells and that the resultant BiFC signal localizes to the cell periphery. Figure6 shows an image that is representative of six independent experiments. Interaction assays of GPA1 with the PLDm1 residue substitution mutant in the same six experiments were devoid of BiFC signal, confirming the importance of the PLDα1 EKF/DRY motif in G-protein interaction (Figure6c,d). We present the GPA1–PLDα1 interaction as a maximum projection of Z-stack images through an isolated cell to show the subcellular localization of the interaction (Figure6b). Population-level images are presented in Figure S9 which shows low-magnification overviews of an additional four experiments in which GPA1–PLDα1 interaction produced extensive BiFC signal. Parallel tests of GPA1–PLDm1 interaction did not show any BiFC signal (Figure S9), and the Golgi marker confirmed successful transformation in every case (Figure S10). In total, GPA1 interacted with PLDα1 in 100% of experiments (10/10) but did not interact with PLDm1 in any experiments (0/10). PLDα1 and PLDm1 protein abundance was comparable by western analysis (Figure S3a). Notably, careful adherence to low agroinfiltration optical densities (0.005–0.02) was key for PLDα1 and PLDm1 assay optimization, presumably due to the negative consequences of sustained lipase activity; difficulty in working with PLDα1 in vivo has been reported previously (Zhao and Wang, 2013).


Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors.

Gookin TE, Assmann SM - Plant J. (2014)

GPA1 interacts with PLDα1, but not with the PLDm1 (EKF→GAA) residue substitution mutant.(a) The GPA1:NmVen210—X:CVen210 parent vector shows no background BiFC signal even when agroinfiltrated into Nicotiana benthamiana leaves at an optical density of 0.2 and imaged 66 h post-infiltration; maximum projection shows blue XT–Golgi–mTq2 signal marking transformed cells.(b) GPA1:NmVen210 interacts with PLDα1:CVen210; maximum projection shows the signal is only at the cell periphery.(c) GPA1:NmVen210 appears not to interact with PLDm1:CVen210, but the presence of transformed cells cannot be confirmed when p19 is in MCS2.(d) GPA1:NmVen210 does not interact with PLDm1:CVen210; the mTq2 Golgi marker clearly identifies transformed cells, confirming a true negative event even when few cells are transformed.(e) GPA1:NmVen210 interaction with PLDa1::CVen210 occurs in Arabidopsis gpa1-3 mesophyll protoplasts.(f) GPA1:NmVen210 does not interact with PLDm1:CVen210 in Arabidopsis gpa1-3 mesophyll protoplasts.(g) GPA1:NmVen210 also does not interact with PLDm2 in Arabidopsis gpa1-3 mesophyll protoplasts. Agroinfiltration of N. benthamiana at very low optical densities (0.005–0.01) allows interaction assays (b, d) in well-isolated cells 44 h post-infiltration. Arabidopsis protoplasts were imaged 12 h post-transformation. Maximum projections (a) ×20 and (b) ×40 magnifications with digital zoom. Single focal plane images at (c, d) ×40 and (e–g) at ×63 magnifications. Identical mVenus channel parameters for (e–g) allow direct comparison. All scale bars = 20 μm except (c) = 50 μm. Yellow = mVenus BiFC, blue = mTq2 Golgi marker, red = autofluorescence.
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fig06: GPA1 interacts with PLDα1, but not with the PLDm1 (EKF→GAA) residue substitution mutant.(a) The GPA1:NmVen210—X:CVen210 parent vector shows no background BiFC signal even when agroinfiltrated into Nicotiana benthamiana leaves at an optical density of 0.2 and imaged 66 h post-infiltration; maximum projection shows blue XT–Golgi–mTq2 signal marking transformed cells.(b) GPA1:NmVen210 interacts with PLDα1:CVen210; maximum projection shows the signal is only at the cell periphery.(c) GPA1:NmVen210 appears not to interact with PLDm1:CVen210, but the presence of transformed cells cannot be confirmed when p19 is in MCS2.(d) GPA1:NmVen210 does not interact with PLDm1:CVen210; the mTq2 Golgi marker clearly identifies transformed cells, confirming a true negative event even when few cells are transformed.(e) GPA1:NmVen210 interaction with PLDa1::CVen210 occurs in Arabidopsis gpa1-3 mesophyll protoplasts.(f) GPA1:NmVen210 does not interact with PLDm1:CVen210 in Arabidopsis gpa1-3 mesophyll protoplasts.(g) GPA1:NmVen210 also does not interact with PLDm2 in Arabidopsis gpa1-3 mesophyll protoplasts. Agroinfiltration of N. benthamiana at very low optical densities (0.005–0.01) allows interaction assays (b, d) in well-isolated cells 44 h post-infiltration. Arabidopsis protoplasts were imaged 12 h post-transformation. Maximum projections (a) ×20 and (b) ×40 magnifications with digital zoom. Single focal plane images at (c, d) ×40 and (e–g) at ×63 magnifications. Identical mVenus channel parameters for (e–g) allow direct comparison. All scale bars = 20 μm except (c) = 50 μm. Yellow = mVenus BiFC, blue = mTq2 Golgi marker, red = autofluorescence.
Mentions: We cloned Arabidopsis wild-type PLDα1 into MCS3 of our GPA1 parent vector to produce a BiFC construct in the GPA1:NmVen210—PLDα1:CVen210 configuration. Using site-directed mutagenesis, we also created PLDα1 mutant versions containing an EKF→GAA substitution (PLDm1) and a longer EKFRVY→GAASGS substitution (PLDm2) which also destroys part of an adjacent highly conserved hydrophobic patch. Importantly, these single vector constructs all contain an integrated Golgi-localized mTurquoise2 marker (XT-Golgi-mTq2) to specifically identify transformed cells. Agroinfiltration into N. benthamiana leaves showed that GPA1 can interact with wild-type PLDα1 in living plant cells and that the resultant BiFC signal localizes to the cell periphery. Figure6 shows an image that is representative of six independent experiments. Interaction assays of GPA1 with the PLDm1 residue substitution mutant in the same six experiments were devoid of BiFC signal, confirming the importance of the PLDα1 EKF/DRY motif in G-protein interaction (Figure6c,d). We present the GPA1–PLDα1 interaction as a maximum projection of Z-stack images through an isolated cell to show the subcellular localization of the interaction (Figure6b). Population-level images are presented in Figure S9 which shows low-magnification overviews of an additional four experiments in which GPA1–PLDα1 interaction produced extensive BiFC signal. Parallel tests of GPA1–PLDm1 interaction did not show any BiFC signal (Figure S9), and the Golgi marker confirmed successful transformation in every case (Figure S10). In total, GPA1 interacted with PLDα1 in 100% of experiments (10/10) but did not interact with PLDm1 in any experiments (0/10). PLDα1 and PLDm1 protein abundance was comparable by western analysis (Figure S3a). Notably, careful adherence to low agroinfiltration optical densities (0.005–0.02) was key for PLDα1 and PLDm1 assay optimization, presumably due to the negative consequences of sustained lipase activity; difficulty in working with PLDα1 in vivo has been reported previously (Zhao and Wang, 2013).

Bottom Line: Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives.We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells.We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 'DRY' motif abolishes both of these interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Pennsylvania State University, University Park, PA, 16802, USA.

Show MeSH
Related in: MedlinePlus