Subcellular Localization of Interacting Proteins by Bimolecular Fluorescence Complementation in Planta

Citovsky, Vitaly; Lee, Lan‐Ying; Vyas, Shachi; Glick, Efrat; Chen, Min-Huei; Vainstein, Alexander; Gafni, Yedidya; Gelvin, Stanton B.; Tzfira, Tzvi

doi:10.1016/j.jmb.2006.08.017

Cited by 349 publications

(339 citation statements)

References 84 publications

(90 reference statements)

Supporting

Mentioning

329

Contrasting

Unclassified

Order By: Relevance

“…Proof that ATG1a and ATG13a directly interact with each other in planta was provided by colocalization of the two proteins using bimolecular fluorescence complementation (BiFC) of the split yellow fluorescent protein (YFP) (Citovsky et al, 2006). Whereas simultaneous expression of the N-and C-terminal halves of YFP failed to reconstitute intact YFP and generate a fluorescent signal in Arabidopsis leaf protoplasts following transient coexpression, such BiFC fluorescence could be detected by confocal microscopy when ATG1a and ATG13a were fused in either arrangement to the C termini of nYFP or cYFP ( Figure 2C).…”

Section: Atg1 and Atg13 Interact With Each Other In Possible Autophagmentioning

confidence: 99%

“…Full-length coding regions for GFP, GFP-ATG8a, ATG1a-GFP, and GFP-ATG13a were placed under the control of the 35S promoter in the plasmids pMDC43 or pMDC83 (Curtis and Grossniklaus, 2003). For BiFC assays, full-length cDNAs for ATG1a and ATG13a were placed after the N-terminal half of EYFP in the pSAT4-DEST-n(1-174)EYFP-C1 vector or after the C-terminal half of EYFP in the pSAT5-DEST-c(175-end)EYFP-C1 (B) vector through LR reactions, respectively (Citovsky et al, 2006). The purified plasmids were introduced into the protoplasts by polyethylene glycol-mediated transformation (Lee et al, 2003) and observed by fluorescence microscopy 18 h later.…”

Section: Protoplast Isolation and Fluorescence Confocal Microscopymentioning

confidence: 99%

See 1 more Smart Citation

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

et al. 2011

View full text Add to dashboard Cite

Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG1a and ATG13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant's nutritional status.

show abstract

Section: Atg1 and Atg13 Interact With Each Other In Possible Autophagmentioning

confidence: 99%

Section: Protoplast Isolation and Fluorescence Confocal Microscopymentioning

confidence: 99%

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Enzyme interactions were tested in planta using the approach of bimolecular fluorescence complementation (BiFC; Citovsky et al, 2006) by transient expression in isolated maize protoplasts. Protoplasts maintain their tissue specificity and reflect in vivo conditions (Faraco et al, 2011;Denecke et al, 2012) and therefore are valuable for examining enzyme interactions.…”

Section: Testing Plastid-localized Interactions Of Partner Hydroxylasesmentioning

confidence: 99%

“…For cloning into pSAT-2236 [pSAT4(A)-nEYFP-N1], a full-length cDNA without the stop codon of the CYP97A4 open reading frame was amplified from pRT-A4 using primers 2455 and 2426 (Citovsky et al, 2006). CYP97C2 was amplified from pGEMT-C2 using primers 3025 and 3026.…”

Section: Psat Constructs Used For Bifc Experimentsmentioning

confidence: 99%

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

et al. 2012

View full text Add to dashboard Cite

Plant carotenoids play essential roles in photosynthesis, photoprotection, and as precursors to apocarotenoids. The plastidlocalized carotenoid biosynthetic pathway is mediated by well-defined nucleus-encoded enzymes. However, there is a major gap in understanding the nature of protein interactions and pathway complexes needed to mediate carotenogenesis. In this study, we focused on carotene ring hydroxylation, which is performed by two structurally distinct classes of enzymes, the P450 CYP97A and CYP97C hydroxylases and the nonheme diiron HYD enzymes. The CYP97A and HYD enzymes both function in the hydroxylation of b-rings in carotenes, but we show that they are not functionally interchangeable. The formation of lutein, which involves hydroxylation of both b-and «-rings, was shown to require the coexpression of CYP97A and CYP97C enzymes. These enzymes were also demonstrated to interact in vivo and in vitro, as determined using bimolecular fluorescence complementation and a pull-down assay, respectively. We discuss the role of specific hydroxylase enzyme interactions in promoting pathway flux and preventing the formation of pathway dead ends. These findings will facilitate efforts to manipulate carotenoid content and composition for improving plant adaptation to climate change and/or for enhancing nutritionally important carotenoids in food crops.

show abstract

“…Moreover combination of different fluorescent marker proteins with appropriate spectral properties permits covisualization of several viral factors at the same time as well as colocalization of viral and host factors (Caplan et al, 2008;Martin et al, 2009;Wei et al, 2010aWei et al, , 2010b. Techniques such as fluorescence resonance energy transfer and bimolecular fluorescence complementation, also based on the use of fluorescent proteins, reveal proteinprotein interactions (Citovsky et al, 2006;Piston and Kremers, 2007;Lalonde et al, 2008). Recently, a new fluorescent marker protein (iLOV), derived from a domain of Arabidopsis (Arabidopsis thaliana) blue light receptor phototropin, was shown to offer some advantages over the GFP-derived markers as a reporter of plant virus infection and movement and when fused to proteins, mainly based on its smaller size (Chapman et al, 2008).…”

mentioning

confidence: 99%

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

et al. 2012

View full text Add to dashboard Cite

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

show abstract

Subcellular Localization of Interacting Proteins by Bimolecular Fluorescence Complementation in Planta

Cited by 349 publications

References 84 publications

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

Contact Info

Product

Resources

About