mouse 1 phosphatidylinositol 4 5 bisphosphate diesterase delta plcd1  (Addgene inc)

Journal: bioRxiv

Article Title: New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

doi: 10.1101/2024.09.06.611683

Figure Lengend Snippet: Expression of YFP-PI3P, YFP-PI4P, YFP-PI5P, YFP-PI(4,5)P 2 and YFP-PI(3,5)P 2 biosensors in chloroplasts of tobacco and identification of their sub-organelle distribution. A. The collection of biosensors is constructed to detect the different PPIs individually. Each specific biosensor consists of a chloroplast transit peptide and a fusion between lipid-binding domains that interacts specifically with each PPI and a yellow fluorescent protein (YFP), mCherry or mEOS4b respectively. B. scheme representing the distribution pattern of PPI biosensors in chloroplast with or without binding to specific PPIs. The upper panel represents a lack of binding of the PPI biosensor with diffuse localization of PPI biosensors in the chloroplast. Lower panel represents the binding of PPI biosensors to PPI-containing membranes with distinct localization of PPI biosensors. C. Confocal images of tobacco leaves transiently expressing PPI biosensors in the chloroplasts. Lacking the binding of the PI3P and PI5P biosensors results in diffuse localization of mEOS4b-PI3P and YFP-PI5P biosensors in the chloroplast. The binding of YFP-PI4P, YFP-PI(4,5)P2 and YFP-PI(3,5)P2 biosensors results in distinct punctate localization of PI biosensors indicating substrate recognition.

Article Snippet: In brief, the coding sequence of the lipid binding domains of the pleckstrin homology (PH) domain of human ARHGEF9 for PI5P binding, switch-associated protein 70 PH domain of human differentially expressed in FDCP6 homolog guanidine exchange factor DEF6 for PI3,4P detection, the PH domain of human rho guanidine nucleotide exchange factor 7 (ARHGEF7) for PI3,5P detection , the four phosphate adaptor protein 1 and 2 (PH) domain of rat FAPP1 for PI4P detection, the PH domain of mouse 1-phosphatidylinositol-4,5-bisphosphate diesterase delta (Plcd1) for PI4,5P detection and putative phosphatidylinositol 3-phosphate binding site encoded by the FYVE domain from human hepatocyte growth factor related tyrosine kinase substrate (HGS11) ( ) was codon optimized for expression in Arabidopsis were codon optimized and synthesized with recombination sites (sequences in supplement) to be integrated into pDK366 containing cTP_YFP, cTP_mCherry or cTP_mEOS4b fusion proteins under the control of the 35S promotor by InFusion clonng., mCherry-FKBP-PTEN was a gift from Gerry Hammond (Addgene plasmid # 116866; http://n2t.net/addgene:116866 ; RRID:Addgene_116866), SAC7/RHD4 (At3g51460) was amplified from Arabidopsis thaliana cDNA. dOCRL was used as codon optimized synthetic DNA of the sequence publishes in .

Techniques: Expressing, Construct, Binding Assay

Journal: bioRxiv

Article Title: New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

doi: 10.1101/2024.09.06.611683

Figure Lengend Snippet: Sub-organelle distribution pattern of mEOS4b-PI3P, mEOS4b-PI4P and YFP-PI(4,5)P 2 biosensors in the presence of the different PPI phosphatases in the chloroplasts. A. The illustration shows a simplified view of the phosphatase activity of PTEN, SAC7, and dOCRL in chloroplasts. B. Distribution pattern of mEOS4b-PI4P biosensors in the presence of phosphatase, SAC7, in the chloroplasts of transiently expressing tobacco. C. Distribution pattern YFP-PI(4,5)P 2 biosensors without and in the presence of presence of PI5P and PI3P phosphatases, dOCRL-mCherry and PTEN-mCherry in the chloroplasts of tobacco. D. Distribution pattern of PI3P biosensors in the presence of PI3P and PI4P phosphatases, PTEN-mCherry and dOCRL-mCherry in the chloroplasts of tobacco. Fluorescence signals were visualized in tobacco leaves co-expressing PPI biosensors and phosphatases (as indicated) by fluorescence confocal microscopy.

Article Snippet: In brief, the coding sequence of the lipid binding domains of the pleckstrin homology (PH) domain of human ARHGEF9 for PI5P binding, switch-associated protein 70 PH domain of human differentially expressed in FDCP6 homolog guanidine exchange factor DEF6 for PI3,4P detection, the PH domain of human rho guanidine nucleotide exchange factor 7 (ARHGEF7) for PI3,5P detection , the four phosphate adaptor protein 1 and 2 (PH) domain of rat FAPP1 for PI4P detection, the PH domain of mouse 1-phosphatidylinositol-4,5-bisphosphate diesterase delta (Plcd1) for PI4,5P detection and putative phosphatidylinositol 3-phosphate binding site encoded by the FYVE domain from human hepatocyte growth factor related tyrosine kinase substrate (HGS11) ( ) was codon optimized for expression in Arabidopsis were codon optimized and synthesized with recombination sites (sequences in supplement) to be integrated into pDK366 containing cTP_YFP, cTP_mCherry or cTP_mEOS4b fusion proteins under the control of the 35S promotor by InFusion clonng., mCherry-FKBP-PTEN was a gift from Gerry Hammond (Addgene plasmid # 116866; http://n2t.net/addgene:116866 ; RRID:Addgene_116866), SAC7/RHD4 (At3g51460) was amplified from Arabidopsis thaliana cDNA. dOCRL was used as codon optimized synthetic DNA of the sequence publishes in .

Techniques: Activity Assay, Expressing, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

doi: 10.1101/2024.09.06.611683

Figure Lengend Snippet: Co-localization of VIPP1 with cTP-mEOS4b-PI3P (FYVE) biosensor and its differential sub-organelle localization in the presence of the PtdIns3P phosphatase, PTEN in the chloroplasts. A. Confocal images of tobacco leaves transiently expressing mEOS4b-PI3P (FYVE) biosensor in the presence and absence of VIPP1-CFP (middle and upper panel). Middle panels show colocalization of mEOS4b-PI3P biosensor with VIPP1-CFP in the chloroplasts. Lower panel shows different localization of mEOS4b-PI(4,5)P 2 biosensor and VIPP1-CFP in the chloroplasts. B. Sub-organelle distribution pattern of VIPP1-CFP in the presence of PI3P phosphatases, PTEN in the chloroplasts. C. Plant lipid based GUVs (magenta) with and without PI3P in the presence or absence of recombinant mEOS4b-VIPP1 (green).

Article Snippet: In brief, the coding sequence of the lipid binding domains of the pleckstrin homology (PH) domain of human ARHGEF9 for PI5P binding, switch-associated protein 70 PH domain of human differentially expressed in FDCP6 homolog guanidine exchange factor DEF6 for PI3,4P detection, the PH domain of human rho guanidine nucleotide exchange factor 7 (ARHGEF7) for PI3,5P detection , the four phosphate adaptor protein 1 and 2 (PH) domain of rat FAPP1 for PI4P detection, the PH domain of mouse 1-phosphatidylinositol-4,5-bisphosphate diesterase delta (Plcd1) for PI4,5P detection and putative phosphatidylinositol 3-phosphate binding site encoded by the FYVE domain from human hepatocyte growth factor related tyrosine kinase substrate (HGS11) ( ) was codon optimized for expression in Arabidopsis were codon optimized and synthesized with recombination sites (sequences in supplement) to be integrated into pDK366 containing cTP_YFP, cTP_mCherry or cTP_mEOS4b fusion proteins under the control of the 35S promotor by InFusion clonng., mCherry-FKBP-PTEN was a gift from Gerry Hammond (Addgene plasmid # 116866; http://n2t.net/addgene:116866 ; RRID:Addgene_116866), SAC7/RHD4 (At3g51460) was amplified from Arabidopsis thaliana cDNA. dOCRL was used as codon optimized synthetic DNA of the sequence publishes in .

Techniques: Expressing, Recombinant

Journal: bioRxiv

Article Title: New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

doi: 10.1101/2024.09.06.611683

Figure Lengend Snippet: Sub-organelle distribution pattern of YFP-PI4P, YFP-PI5P, YFP-PI(4,5)P 2 and YFP-PI(3,5)P 2 biosensors as well as cTP-GFP in the chloroplast of Arabidopsis seedlings under control and heat stress (HS) conditions. A and B. Confocal images of 10-days-old Arabidopsis seedlings stably expressing cTP-YFP-PI4P , cTP-YFP-PI5P , cTP-YFP-PI(4,5)P 2 and c TP-YFP-PI(3,5)P 2 biosensors. (A) control and (B) HS (at 40°C for 30 minutes). The diagrams at the right represent PIP flux upon control and HS. C. Immunoblot analysis of cTP-GFP, YFP-PI4P, YFP-PI5P, YFP-PI(4,5)P and YFP-PI(3,5)P 2 proteins under control and HS conditions using GFP antibody.

Article Snippet: In brief, the coding sequence of the lipid binding domains of the pleckstrin homology (PH) domain of human ARHGEF9 for PI5P binding, switch-associated protein 70 PH domain of human differentially expressed in FDCP6 homolog guanidine exchange factor DEF6 for PI3,4P detection, the PH domain of human rho guanidine nucleotide exchange factor 7 (ARHGEF7) for PI3,5P detection , the four phosphate adaptor protein 1 and 2 (PH) domain of rat FAPP1 for PI4P detection, the PH domain of mouse 1-phosphatidylinositol-4,5-bisphosphate diesterase delta (Plcd1) for PI4,5P detection and putative phosphatidylinositol 3-phosphate binding site encoded by the FYVE domain from human hepatocyte growth factor related tyrosine kinase substrate (HGS11) ( ) was codon optimized for expression in Arabidopsis were codon optimized and synthesized with recombination sites (sequences in supplement) to be integrated into pDK366 containing cTP_YFP, cTP_mCherry or cTP_mEOS4b fusion proteins under the control of the 35S promotor by InFusion clonng., mCherry-FKBP-PTEN was a gift from Gerry Hammond (Addgene plasmid # 116866; http://n2t.net/addgene:116866 ; RRID:Addgene_116866), SAC7/RHD4 (At3g51460) was amplified from Arabidopsis thaliana cDNA. dOCRL was used as codon optimized synthetic DNA of the sequence publishes in .

Techniques: Control, Stable Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

doi: 10.1101/2024.09.06.611683

Figure Lengend Snippet: Perturbation of PIP abundance leads to drought sensitivity in Arabidopsis plants expressing PI4P phosphatase (SAC7), PI3P phosphatase (PTEN), and PI5P phosphatase (dOCRL). A. Sub-organelle distribution pattern of mEOS4b-PI3P, YFP-PI4P, YFP-PI5P, YFP-PI(4,5)P 2 and YFP-PI(3,5)P 2 biosensors in the chloroplast of Arabidopsis seedlings under control and drought stress conditions. Confocal images of 10-days-old Arabidopsis seedlings stably expressing cTP-mEOS4b-PI3P , cTP-YFP-PI4P , cTP-YFP-PI5P , cTP-YFP-PI(4,5)P 2 and c TP-YFP-PI(3,5)P 2 biosensors. For drought stress leaf samples from plants growing on petri dishes were detached and kept for 30 minutes at RT prior to imaging. B. Phenotypes of WT, cTP-PTEN.OX (left), cTP-SAC7.OX (middle) and cTP-DOCRL.OX (right) plants under control condition (well watered; upper panel) and after drought stress (middle panel). Seedlings were grown for 4 weeks in soil under a diurnal cycle of 16 h light (120 μE m −2 s −1 ) and 8 h dark at 22 °C. Drought stress was applied by withholding water for 10-14 days. The lower panel indicates Fv/Fm measurements of drought-stressed cTP-PTEN.OX (left) and cTP-SAC7.OX (right) plants. B. TEM images of Arabidopsis WT and cTP-PTEN.OX (upper images). TEM images of Arabidopsis WT and plants expressing cTP-PTEN.OX following drought treatments (lower images).

Article Snippet: In brief, the coding sequence of the lipid binding domains of the pleckstrin homology (PH) domain of human ARHGEF9 for PI5P binding, switch-associated protein 70 PH domain of human differentially expressed in FDCP6 homolog guanidine exchange factor DEF6 for PI3,4P detection, the PH domain of human rho guanidine nucleotide exchange factor 7 (ARHGEF7) for PI3,5P detection , the four phosphate adaptor protein 1 and 2 (PH) domain of rat FAPP1 for PI4P detection, the PH domain of mouse 1-phosphatidylinositol-4,5-bisphosphate diesterase delta (Plcd1) for PI4,5P detection and putative phosphatidylinositol 3-phosphate binding site encoded by the FYVE domain from human hepatocyte growth factor related tyrosine kinase substrate (HGS11) ( ) was codon optimized for expression in Arabidopsis were codon optimized and synthesized with recombination sites (sequences in supplement) to be integrated into pDK366 containing cTP_YFP, cTP_mCherry or cTP_mEOS4b fusion proteins under the control of the 35S promotor by InFusion clonng., mCherry-FKBP-PTEN was a gift from Gerry Hammond (Addgene plasmid # 116866; http://n2t.net/addgene:116866 ; RRID:Addgene_116866), SAC7/RHD4 (At3g51460) was amplified from Arabidopsis thaliana cDNA. dOCRL was used as codon optimized synthetic DNA of the sequence publishes in .

Techniques: Expressing, Control, Stable Transfection, Imaging