pentr™/u6 rnai entry vector  (Thermo Fisher)

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Simplified model for PP-InsP synthesis, degradation, and regulation of PSR genes. IPK1 synthesizes InsP 6 , ITPK enzymes synthesize InsP 7 , and VIP enzymes synthesize InsP 8 in Arabidopsis . Transgenic expression of DDP1 ( S. cerevisiae ) or NUDIX13 ( Arabidopsis ) is predicted to hydrolyze InsP 7 and InsP 8 in planta. It is important to note that this is a simplified figure and does not address specific InsP isomers or the newly discovered isomers of InsP 7 ( ; ). Under P i -deplete conditions, dimerized PHR binds to the P1BS promoter and upregulates PSR genes (“PSR ON”) . Under P i -replete conditions, InsP 8 is assumed to be the controller to turn the PSR off. InsP 8 binds to SPX and this SPX:InsP 8 complex disrupts the PHR dimer and inhibits PHR-mediated transcription of PSR genes.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing NUDIX13 was recombined with Gateway destination vector pDEST15 (Invitrogen, N-terminus GST) using the Gateway LR Clonase II kit (Invitrogen Corp., Carlsbad, CA, USA), and GST-NUDIX13 sequence was verified as above.

Techniques: Transgenic Assay, Expressing

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Characterization of NUDIX13 OX transgenics. A) Immunoblot of 3-wk-old leaf tissue from WT and selected NUDIX13 OX transgenics. Ponceau staining shows Rubisco accumulation in all plants as a positive control (∼50 to 56 kDa). B) Average rosette diameter over time. Each point represents n = 3 independent experiments with over 12 plants per genotype per experiment; error bars show Sd . Different letters indicate statistically significant means (Tukey honestly significant difference (HSD) test, α = 0.05). C) Arabidopsis rosette growth over the course of 5 wk, each image representative of n = 4 to 5 independent experiments containing 12 or more plants per genotype. Images were digitally extracted for comparison. Scale bar = 1 cm. D) Close-up view of yellowing cauline leaves. White arrowheads indicate yellowing cauline leaves. Scale bar = 1 cm. E) HPLC analysis of WT and NUDIX13 OX transgenics, n = 3. See for additional profiles. F) Total InsP 7 , G) total InsP 8 , H) InsP 7 /InsP 6 , and I) InsP 8 /InsP 6 ratios. Asterisks show significant differences from WT; analyzed using Student's t -test; * P < 0.05, error bars show Sd of n = 3. J , K) Leaf P i accumulation in soil-grown plants from 3 J) and 6 wk K) of growth. Analyzed using Student's t -test; n = 2 to 3 independent experiments; * P < 0.05, error bars show Sd .

Article Snippet: Gateway pENTR/D-TOPO entry vector containing NUDIX13 was recombined with Gateway destination vector pDEST15 (Invitrogen, N-terminus GST) using the Gateway LR Clonase II kit (Invitrogen Corp., Carlsbad, CA, USA), and GST-NUDIX13 sequence was verified as above.

Techniques: Western Blot, Staining, Positive Control, Comparison

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: The average percent of total, individual InsP species compared to the total WT pool expressed as a percentage Averages for NUDIX13 OX were taken from all replicates shown in Fig. 4 and Supplementary Fig. 2 and nudix13-1 from Supplementary Fig. S3 ; n = 3 biological replicates per genotype

Article Snippet: Gateway pENTR/D-TOPO entry vector containing NUDIX13 was recombined with Gateway destination vector pDEST15 (Invitrogen, N-terminus GST) using the Gateway LR Clonase II kit (Invitrogen Corp., Carlsbad, CA, USA), and GST-NUDIX13 sequence was verified as above.

Techniques:

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: In vitro phosphatase assays with purified recombinant DDP1-GST and NUDIX13-GST. A) Purified DDP1-GST, NUDIX13-GST, or GST alone (negative control) was incubated with polyP (Lanes 1 to 4), Ap 5 A (Lanes 5 to 8), or Ap 4 A (Lanes 9 to 11) for 1.5 h at 37 °C, and then the reactions were resolved by PAGE and stained with Toluidine blue. Bands corresponding to Orange G (loading dye), Ap 5 A, and Ap 4 A are indicated on the right; undigested polyP appears ladderlike. Reactions in the absence (−) or presence (+) of enzyme or control GST protein are indicated in the table. B to D) DDP1-GST, NUDIX13-GST, or GST alone (negative control) was incubated with radiolabeled InsP 8 B) or InsP 7 D) for 1.5 h at 37 °C, and then the reaction products were resolved by HPLC and measured by liquid scintillation counting. Representative chromatograms with peaks corresponding to InsP 6 , InsP 7 , or InsP 8 are shown. Reactions that lacked enzyme or GST control protein (traces labeled “InsP 7 / 8 substrate”) show the starting substrate in each reaction. C) Percentage of InsP 8 hydrolyzed by each enzyme ( n = 3); error bars show Sd . D) HPLC analyses of enzymatically synthesized InsP 7 substrate incubated with DDP1 or NUDIX13, or without enzyme, n = 1.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing NUDIX13 was recombined with Gateway destination vector pDEST15 (Invitrogen, N-terminus GST) using the Gateway LR Clonase II kit (Invitrogen Corp., Carlsbad, CA, USA), and GST-NUDIX13 sequence was verified as above.

Techniques: In Vitro, Purification, Recombinant, Negative Control, Incubation, Staining, Control, Labeling, Synthesized

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Confocal imaging of DDP1-GFP and NUDIX13-GFP expression in Arabidopsis and N. benthamiana . A , B) Arabidopsis DDP1-I leaves and C) roots, D) DDP1-A leaves, E) NUDIX13-H leaves, and F) NUDIX13-O leaves. G) CaMV35S:DDP1-GFP and J) CaMV35S:NUDIX13-GFP transient expression in N. benthamiana leaves 48-h postinfiltration. N. benthamiana leaves coinfiltrated with DDP1-GFP G to I) or NUDIX13-GFP J to L) with unconjugated mCherry. DDP1-GFP and NUDIX13 are shown in green, and unconjugated mCherry is shown in magenta. All images are presented as maximum intensity projections from confocal Z -stack optical sections. All scale bars = 50 µ m.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing NUDIX13 was recombined with Gateway destination vector pDEST15 (Invitrogen, N-terminus GST) using the Gateway LR Clonase II kit (Invitrogen Corp., Carlsbad, CA, USA), and GST-NUDIX13 sequence was verified as above.

Techniques: Imaging, Expressing

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Simplified model for PP-InsP synthesis, degradation, and regulation of PSR genes. IPK1 synthesizes InsP 6 , ITPK enzymes synthesize InsP 7 , and VIP enzymes synthesize InsP 8 in Arabidopsis . Transgenic expression of DDP1 ( S. cerevisiae ) or NUDIX13 ( Arabidopsis ) is predicted to hydrolyze InsP 7 and InsP 8 in planta. It is important to note that this is a simplified figure and does not address specific InsP isomers or the newly discovered isomers of InsP 7 ( ; ). Under P i -deplete conditions, dimerized PHR binds to the P1BS promoter and upregulates PSR genes (“PSR ON”) . Under P i -replete conditions, InsP 8 is assumed to be the controller to turn the PSR off. InsP 8 binds to SPX and this SPX:InsP 8 complex disrupts the PHR dimer and inhibits PHR-mediated transcription of PSR genes.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: Transgenic Assay, Expressing

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Characterization and comparison of DDP1 OX transgenics. A) Arabidopsis rosette growth over the course of 6 wk, each image representative of n = 3 to 4 independent experiments containing 12 or more plants per genotype. Images were digitally extracted for comparison. Scale bar = 1 cm. B) Close-up view of aborting siliques in a 5-wk-old DDP1-I plant. The three white arrowheads at the top of the image indicate aborting siliques, and the three yellow arrowheads at the bottom of the image mark yellowing cauline leaves. Scale bar = 1 cm. C) Average rosette diameter over time. Each point represents n = 3 independent experiments with over 12 plants per genotype per experiment; error bars show Sd . Different letters indicate statistically significant means (Tukey honestly significant difference (HSD) test, α = 0.05). D) Immunoblot of 4-wk-old leaf tissue from WT and selected DDP1 OX transgenics. Ponceau staining shows Rubisco accumulation in all plants as a positive control (∼50 to 56 kDa). E) WT and DDP1 OX transgenics grown on vermiculite containing 0.5× MS media with 10 μ m KH 2 PO 4 (deplete P i ) or 1 m m KH 2 PO 4 (replete P i ) after 35 d. DDP1-I plants did not accumulate lesions under deplete P i and resemble WT plants on deplete P i . F) Rosette diameter measurements of WT and DDP1-I grown under deplete and replete P i . Each point represents an individual plant measurement from n = 2 independent experiments; over 35 plants per genotype and condition; error bars show Sd . Different letters indicate statistically significant means (Tukey HSD, α = 0.05). G) DDP1-I transgenics after 50 d of growth on vermiculite. DDP1-I lines grown on deplete P i did not have aborted siliques.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: Comparison, Western Blot, Staining, Positive Control

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: PP-InsP profiling, P i accumulation, and PSR gene expression in DDP1 OX transgenics. A) WT and DDP1 OX transgenics were grown for 14 d on semisolid 0.5× MS media with 0.2% agar then 100 μ Ci [ 3 H]- myo- inositol was added for 4 d. All InsPs were extracted, separated using anion exchange HPLC, and data were analyzed as described in the “Materials and methods” section. These InsP profiles are representative of 2 to 3 independent replicates per genotype; see for all profiles. B) InsP 7 /InsP 6 and C) InsP 8 /InsP 6 ratios. Asterisks show significant differences from WT; analyzed using Student's t -test; * P < 0.05, error bars show Sd of n = 2 to 3. D) Leaf P i accumulation in soil-grown plants from 3 to 7 wk of growth. Each point represents pooled plant tissue from n = 2 to 4 independent experiments; error bars show Sd . Different letters indicate statistically significant means (Tukey honestly significant difference (HSD) test, α = 0.05). E to G) WT and DDP1 OX transgenics were grown for 100 d in hydroponic tracks in a greenhouse. E) Amount of P accumulated in shoot dry mass of WT and DDP1 OX transgenics. Each point represents the average of one track of n = 9 to 14 plants; error bars show Sd (Tukey HSD, α = 0.05). F) The amount of total P accumulated per individual plant regardless of total mass (WT n = 45; DDP1 Ox n = 49) and G) plants weighing 200 mg or less (WT n = 15; DDP1 Ox n = 29); (Tukey HSD, α = 0.05). H) PSR gene expression (relative to WT) and I) P i accumulation in whole seedlings grown for 10 d grown on media plates. Error bars denote Sd of n = 3 independent experiments. Different letters indicate statistically significant means (Tukey HSD, α = 0.05).

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: Expressing

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Average percent of the total counts per minute for each InsP as a percentage compared to the total WT pool for the respective InsP species

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques:

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: In vitro phosphatase assays with purified recombinant DDP1-GST and NUDIX13-GST. A) Purified DDP1-GST, NUDIX13-GST, or GST alone (negative control) was incubated with polyP (Lanes 1 to 4), Ap 5 A (Lanes 5 to 8), or Ap 4 A (Lanes 9 to 11) for 1.5 h at 37 °C, and then the reactions were resolved by PAGE and stained with Toluidine blue. Bands corresponding to Orange G (loading dye), Ap 5 A, and Ap 4 A are indicated on the right; undigested polyP appears ladderlike. Reactions in the absence (−) or presence (+) of enzyme or control GST protein are indicated in the table. B to D) DDP1-GST, NUDIX13-GST, or GST alone (negative control) was incubated with radiolabeled InsP 8 B) or InsP 7 D) for 1.5 h at 37 °C, and then the reaction products were resolved by HPLC and measured by liquid scintillation counting. Representative chromatograms with peaks corresponding to InsP 6 , InsP 7 , or InsP 8 are shown. Reactions that lacked enzyme or GST control protein (traces labeled “InsP 7 / 8 substrate”) show the starting substrate in each reaction. C) Percentage of InsP 8 hydrolyzed by each enzyme ( n = 3); error bars show Sd . D) HPLC analyses of enzymatically synthesized InsP 7 substrate incubated with DDP1 or NUDIX13, or without enzyme, n = 1.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: In Vitro, Purification, Recombinant, Negative Control, Incubation, Staining, Control, Labeling, Synthesized

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Confocal imaging of DDP1-GFP and NUDIX13-GFP expression in Arabidopsis and N. benthamiana . A , B) Arabidopsis DDP1-I leaves and C) roots, D) DDP1-A leaves, E) NUDIX13-H leaves, and F) NUDIX13-O leaves. G) CaMV35S:DDP1-GFP and J) CaMV35S:NUDIX13-GFP transient expression in N. benthamiana leaves 48-h postinfiltration. N. benthamiana leaves coinfiltrated with DDP1-GFP G to I) or NUDIX13-GFP J to L) with unconjugated mCherry. DDP1-GFP and NUDIX13 are shown in green, and unconjugated mCherry is shown in magenta. All images are presented as maximum intensity projections from confocal Z -stack optical sections. All scale bars = 50 µ m.

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: Imaging, Expressing

Journal: Plant Physiology

Article Title: Using native and synthetic genes to disrupt inositol pyrophosphates and phosphate accumulation in plants

doi: 10.1093/plphys/kiae582

Figure Lengend Snippet: Characterization of pennycress DDP1 OX transgenics. A) Physiology of WT pennycress and 2 independent heterozygous DDP1 OX transgenics lines after 5 wk of growth. B) Close-up view of DDP1-B leaf tip necrosis and chlorosis after 7 wk of growth on soil. White arrowheads mark yellowing of leaf tips. C) Immunoblot of 6-wk-old leaf tissue from WT and heterozygous pennycress DDP1 OX lines. Ponceau staining shows Rubisco accumulation in all plants as a positive control (∼50 to 56 kDa). D) Stable YFP-DDP1 expression in pennycress DDP1-B leaves. Scale bar = 50 µ m. E) Shoot P i accumulation in 6- to 7-wk-old pennycress leaf tissue. Error bars denote Sd of n = 3 independent experiments; each point represents leaf tissue of an individual heterozygous plant. Different letters indicate statistically significant means (Tukey honestly significant difference (HSD) test, α = 0.05).

Article Snippet: Gateway pENTR/D-TOPO entry vector containing DDP1 was recombined with Gateway destination vector pDEST15 (N-terminus GST; Invitrogen, Carlsbad, CA, USA) using the Gateway LR Clonase II kit (Invitrogen).

Techniques: Western Blot, Staining, Positive Control, Expressing