2in1 Vectors Improve In Planta BiFC and FRET Analyses

Mehlhorn, Dietmar Gerald; Wallmeroth, Niklas; Berendzen, Kenneth W.; Grefen, Christopher

doi:10.1007/978-1-4939-7389-7_11

Cited by 28 publications

(27 citation statements)

References 23 publications

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“…Agrobacterium-mediated transformation of leaves of 4-week-old N. benthamiana plants with the BiFC constructs described herein (see "Plant Expression Constructs") was performed as described previously (Mehlhorn et al, 2018, with the following changes: 4 mL of overnight bacterial cultures was used to inoculate 16 mL of fresh media. The subcultures were grown to an OD 600 5 0.8 and were centrifuged at 8000g for 20 min.…”

Section: Bifc Experimentsmentioning

confidence: 99%

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function

et al. 2019

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The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in attrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

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Section: Bifc Experimentsmentioning

confidence: 99%

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function

et al. 2019

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“…Agrobacterium-mediated transient transformation of 4-6 week old N. benthamiana leaves with 2in1 369 rBiFC constructs as described previously [71]. Fluorescence intensities were recorded 36h post 370 infiltration for approximately 30 images per construct as described in [49].…”

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confidence: 99%

Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

Denninger

Reichelt

Schmidt

et al. 2019

Preprint

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35Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil 36 volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch 37 from cell elongation to tip growth through polarization of the growth machinery to a pre-defined root 38 hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain 39 resembles the establishment of cell polarity in other eukaryotic systems [1][2][3]. Rho-type GTPases 40 of plants (ROPs) are among the first molecular determinants of the RHID [4, 5] and later play a 41 central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the 42 RHID in the cell [7][8][9] or regulate ROP activity [10][11][12][13][14][15][16][17][18]. The molecular players that target ROPs to 43 the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the 44 growth machinery assembly in polarizing hair cells and found that positioning of molecular players 45 and outgrowth are temporally separate processes that are each controlled by specific ROP guanine 46 nucleotide exchange factor (GEFs). A functional analysis of trichoblast-specific GEFs revealed 47 GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, while 48 GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the 49 formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role 50 of GEF3 to serve as a membrane landmark during cell polarization. 51 3 RESULTS AND DISCUSSION 52 53 Temporal analysis of hair cell polarization reveals phased deployment of the tip-growth 54 machinery 55To dissect the process of breaking cellular symmetry and initiating polar growth in plants, we 56 analyzed the gradual assembly of the tip growth machinery in trichoblasts using specific markers 57 for cytoskeletal rearrangement, plasma membrane specialization, cell wall modification, vesicle 58 trafficking, and Rho-GTPase signaling. Using stable transgenic Arabidopsis lines expressing 59 fluorescently labeled versions of these protein markers, we determined the timing of their 60 polarization at the RHID during trichoblast differentiation. As the Arabidopsis root represents a time-61 axis of development from stem cells to mature cells, we numbered the developmental stages (-7 62 to +3) within a cell file, with the last cell before bulging labeled -1 and the first cell after the onset 63 of bulging labeled +1 ( Figure 1A). We used the integral plasma membrane marker GFP-LTI6B [19] 64 as a reference and calculated the polarity index of fluorescently labeled marker proteins to quantify 65 protein accumulation at the RHID (Figure 1B, C). Our survey unveiled a two-phase assembly of 66 the tip growth machinery: an initiation phase, during which the RHID is positioned and predefined, 67 followed by the tip growth phase. Consistent with previous reports [4, 5], mCitrine-labeled (mCit) 68GTPase ROP2 associ...

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“…Construct Generation and Plant Transformation. Most constructs were designed using Gateway technology or the Gateway-compatible cloning system 2in1 (25,28,35). For generation of the reverse-charged mutation of G1IP, three arginine and one lysine residue at positions 9 to 12 were exchanged with glutamic acid residues by site-directed mutagenesis as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

“…G1IP Binds AtGET3a Only in the Presence of AtGET1. To corroborate and expand the analyses of physical interaction of G1IP and G1IP-like with Arabidopsis GET pathway components, we performed rBiFC (25,27,28) and coimmunoprecipitation (co-IP) analyses. Complementation of the YFP signal, a cue for physical interaction, was detected only in samples in which AtGET1 was coexpressed with G1IP or G1IP-like ( Fig.…”

Section: G1ip and Atget1 Share The Same Expression Profile And Subcelmentioning

confidence: 99%

Endoplasmic reticulum membrane receptors of the GET pathway are conserved throughout eukaryotes

Asseck

Mehlhorn

Rivera‐Monroy

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plantArabidopsis thaliana, most components of the pathway were identified throughin silicosequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors ofAtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses withAtGET1, and binds toArabidopsisGET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of otherAtgetlines, its heterologous expression together with the coreceptorAtGET1 rescues growth defects ofΔget1get2yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 inArabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

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2in1 Vectors Improve In Planta BiFC and FRET Analyses

Cited by 28 publications

References 23 publications

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function

Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

Endoplasmic reticulum membrane receptors of the GET pathway are conserved throughout eukaryotes

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