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4. Agrobacterium infiltration of Nicotiana benthamiana leaves. ( a ) A 5-week-old N. benthamiana plant that is appropriate to be infiltrated. The leaves at the optimal developmental stage for infiltration are indicated by arrows. ( b ) Infiltration of the Agrobacterium suspension into the abaxial air space of a tobacco leaf ( see Color Plates ) .
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Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability of cells to respond appropriately to environmental stimuli. However, the study of protein complex formation in living plant cells has remained experimentally difficult and time-consuming and requires sophisticated technical equipment. In...
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Citations
... Bimolecular fluorescence complementation For protein-protein interaction studies the BiFC method with the pSPYCE and pSPYNE vectors was used (Schütze et al., 2009). The coding sequences of the genes were cloned into the vectors, and the vectors were transformed into Agrobacterium tumefaciens. ...
... The coding sequences of the genes were cloned into the vectors, and the vectors were transformed into Agrobacterium tumefaciens. The resulting cultures were used for co-expression in N. benthamiana leaves as previously described (Schütze et al., 2009). ...
It is generally accepted that jasmonate‐ZIM domain (JAZ) repressors act to mediate jasmonate (JA) signaling via CORONATINE‐INSENSITIVE1 (COI1)‐mediated degradation. Here, we report a cryptic signaling cascade where a JAZ repressor, FvJAZ12, mediates multiple signaling inputs via phosphorylation‐modulated subcellular translocation rather than the COI1‐mediated degradation mechanism in strawberry (Fragaria vesca). FvJAZ12 acts to regulate flavor metabolism and defense response, and was found to be the target of FvMPK6, a mitogen‐activated protein kinase that is capable of responding to multiple signal stimuli. FvMPK6 phosphorylates FvJAZ12 at the amino acid residues S179 and T183 adjacent to the PY residues, thereby attenuating its nuclear accumulation and relieving its repression for FvMYC2, which acts to control the expression of lipoxygenase 3 (FvLOX3), an important gene involved in JA biosynthesis and a diverse array of cellular metabolisms. Our data reveal a previously unreported mechanism for JA signaling and decipher a signaling cascade that links multiple signaling inputs with fruit trait development.
... Transient gene expression in N. benthamiana by agroinfiltration was carried out according to a previous described procedure (Schütze et al., 2009). Leaves were harvested 3 days after agroinfiltration for imaging with a Leica SP5 confocal laser scanning microscope or protein content analysis. ...
The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (α) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low-sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.
... This method has been applied frequently for fluorescence microscopy: Multiple proteins have been localized this way, for instance V-ATPase subunits and malate [3,4]. Transient expression enabled the quantitative in vivo visualization of protein-protein interactions by bimolecular fluorescence complementation or Förster resonance energy transfer [4,5]. Transactivation of promoters by transcription factors has been addressed by using protoplasts, too [6]. ...
Plant cells are omnipotent and breeding of new varieties can be achieved by protoplast fusion. Such fusions can be achieved by treatment with poly(ethylene glycol) or by applying an electric field. Microfluidic devices allow for controlled conditions and targeted manipulation of small batches of cells down to single-cell analysis. To provide controlled conditions for protoplast fusions and achieve high reproducibility, we developed and characterized a microfluidic device to reliably trap some Arabidopsis thaliana protoplasts and induced cell fusion by controlled addition of poly(ethylene glycol) (PEG, with a molecular weight of 6000). Experiments were conducted to determine the survival rate of isolated protoplasts in our microfluidic system. Afterward, PEG-induced fusion was studied. Our results indicate that the following fusion parameters had a significant impact on the fusion efficiency and duration: PEG concentration, osmolality of solution and flow velocity. A PEG concentration below 10% led to only partial fusion. The osmolality of the PEG fusion solution was found to strongly impact the fusion process; complete fusion of two source cells sufficiently took part in slightly hyper-osmotic solutions, whereas iso-osmotic solutions led to only partial fusion at a 20% PEG concentration. We observed accelerated fusion for higher fluid velocities. Until this study, it was common sense that fusion is one-directional, i.e., once two cells are fused into one cell, they stay fused. Here, we present for the first time the reversible fusion of protoplasts. Our microfluidic device paves the way to a deeper understanding of the kinetics and processes of cell fusion.
... The full-length or segmented cDNA sequences of VvCEB1 and VvDELLA2 were cloned into pSPYNE or pSPYCE vectors to construct fusions expressing YFP N and YFP C (Supplementary Table S2). The fusion expression vectors were transformed into Agrobacterium tumefaciens and the mixture of two different plasmids of equal volume was transformed instantaneously into N. benthamiana leaves, as described by Schutze et al. [53]. The injected N. benthamiana plants were cultured for 3 days at 28 • C for 8-hour light/16-hour darkness conditions, and the fluorescence of green fluorescent protein (GFP) was observed in the area of N. benthamiana leaves injected with Agrobacterium using a confocal microscope (Olympus Fluoview FV1000). ...
Exogenous gibberellin treatment can promote early growth of grape fruit, but the underlying regulatory mechanisms are not well understood. Here, we show that VvDELLA2 directly regulates the activity of the VvCEB1 transcription factor, a key regulator in the control of cell expansion in grape fruit. Our results show that VvCEB1 binds directly to the promoters of cell expansion-related genes in grape fruit and acts as a transcriptional activator, while VvDELLA2 blocks VvCEB1 function by binding to its activating structural domain. The exogenous gibberellin treatment relieved this inhibition by promoting the degradation of VvDELLA2 protein, thus, allowing VvCEB1 to transcriptionally activate the expression of cell expansion-related genes. In conclusion, we conclude that exogenous GA3 treatment regulates early fruit expansion by affecting the VvDELLA-VvCEB1 interaction in grape fruit development.
... Bimolecular fluorescence complementation (BiFC) is an in vivo method for studying protein-protein interactions in plant cells. The BiFC technique is based on the restoration of fluorescent signals by two non-fluorescent fragments of the yellow fluorescent protein (YFP) brought together by the association of interacting proteins fused to these fragments (Schutze et al., 2009;Schweiger and Schwenkert, 2014). Here, we also show our protoplast transient gene expression system could be used for protein-protein interaction studies in passion fruit, illustrated by the BiFC experiments to study two pairs of fusion proteins: YN-CP and YC-CP. ...
Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method (‘Cotyledon Peeling Method’) for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 10⁷ protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 10⁴ cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.
... There is thus an urgent need to implement and develop new analytical methods for the rapid and robust in planta detection of PPIs and PTMs (Miura, 2018). Commonly used technologies to monitor in vivo PPI in plants include Bimolecular Fluorescence Complementation (BiFC), Förster Resonance Energy Transfer (FRET), and derivatives thereof (Schutze et al., 2009) (Waadt and Kudla, 2008) (Bucherl et al., 2014) (Gadella et al., 1999. In BiFC, two putative interacting proteins are fused to complementary parts of a split fluorescent protein. ...
... In BiFC, two putative interacting proteins are fused to complementary parts of a split fluorescent protein. Only upon interaction, the two parts of the fluorescent reporter can reconstitute and restore the fluorophore (Schutze et al., 2009) (Waadt and Kudla, 2008). The detection of a fluorescent signal is therefore a readout of interaction, making it technically easy to use, which explains its widespread use. ...
Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. The dynamic nature of protein complex formation and post-translational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (SPPIER - Separation of Phases-based Protein Interaction Reporter) and kinase activities (SPARK - Separation of Phases-based Activity Reporter of Kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 (SnRK1) activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other post-translational modifications with unprecedented ease and sensitivity.
... We created the nuclear-localized maker vectors 35S::NLS-RFP and 35S::RH13-GFP for transient transformation of Nicotiana benthamiana. Agrobacterium (GV3101) infiltration of N. benthamiana leaves was performed as described previously (Schütze et al., 2009). The fluorescence in the epidermal cell layer of the lower leaf surface expressing the fusion proteins was observed 1 d after infiltration. ...
Chloroplast is a semi-autonomous organelle with a double membrane structure, and its structural stability is the prerequisite for its correct function. Chloroplast development is regulated by known nuclear-encoded chloroplast proteins or proteins encoded within the chloroplast itself. However, the mechanism of chloroplast development is involved in other organelles remains largely unknown. Here, we report that a nuclear-located DEAD-box RNA helicase 13 (RH13) is essential for chloroplast development in Arabidopsis thaliana. RH13 is widely expressed in tissues and localized to the nucleolus. Homozygous rh13 mutant shows abnormal chloroplast structure and leaf morphogenesis. Proteomic analysis shows that the expression levels of photosynthesis related proteins in chloroplasts are reduced due to loss of RH13. Furthermore, RNA-sequencing and proteomics data reveals that the expression levels of these chloroplast-related genes are decreased which undergo alternative splicing events in rh13 mutant. Taken together, we propose that nucleolus-located RH13 is critical for chloroplast development in Arabidopsis.
... The advancement of genetic modifications and protein labelling led to the development of another binary method which utilises fluorescent protein technology to study PPI in vivo. Similar to Y2H, the BiFC assay operates on the principle of protein reconstitution, but it uses fluorescent proteins such as green fluorescent protein (GFP) [82] (Table 1). BiFC is a popular method in plant PPI studies as it allows easy visualisation of interacting proteins in living cells without altering the chemical properties of the interaction in its subcellular location [83]. ...
Interactomics is a branch of systems biology that deals with the study of protein-protein interactions and how these interactions influence phenotypes. Identifying the interactomes involved during host-pathogen interaction events may bring us a step closer to deciphering the molecular mechanisms underlying plant defence. Here, we conducted a systematic review of plant interactomics studies over the last two decades and found that while a substantial progress has been made in the field, plant-pathogen interactomics remains a less-travelled route. As an effort to facilitate the progress in this field, we provide here a comprehensive research pipeline for an in planta plant-pathogen interactomics study that encompasses the in silico prediction step to the validation step, unconfined to model plants. We also highlight four challenges in plant-pathogen inter-actomics with plausible solution(s) for each.
... The synthetized fragment was cloned into the binary vector pKGWFS7, which fuses the promoter to the eGFP and the β-glucuronidase (GUS) coding sequences. Positive vectors were transformed into Agrobacterium tumefaciens strain GV3101 and selected by using suitable antibiotics.Transient expression in N. benthamiana leaves was assessed according to(Schutze et al., 2009). Briefly, an overnight Agrobacterium culture was collected, resuspended in AS medium [10 mM MgCl2, 10 mM morpholine ethanesulfonic acid (MES) (pH 5.6) and 150μM acetosyringone] and incubated at room temperature for 2 h. ...
In this work, we identified and functionally characterized the R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by abscisic acid and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in total anthocyanin and flavonoid contents that were especially marked in malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for a R2R3-MYB TF, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.
... The full-length cDNA fragment (without stop codon) of AtHAE was amplified from wild-type Col-0 cDNA and cloned into pSPYNE (Schütze et al., 2009) Fluorescence of the epidermal cell layer of the lower leaf surface was examined at 3 dpi. Images were captured using an LSM 880 confocal microscope (Zeiss). ...
Plant‐parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185‐amino‐acid secreted protein, was previously shown to be up‐regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana–H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface‐associated leucine‐rich repeat (LRR) receptor‐like kinase (RLK) involved in signalling the activation of cell wall‐remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA‐LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01–AtHAE interaction may play an important role in sugar beet cyst nematode parasitism. The novel cyst nematode effector 2D01 targets the intracellular kinase domain of the cell surface receptor HAESA to promote parasitism.
