Stromule extension along microtubules coordinated with actin-mediated anchoring guides perinuclear chloroplast movement during innate immunity
- University of Delaware, United States
- University of California, Davis, United States
Figures
Figure 1 with 1 supplement
Chloroplast stromules extend along microtubules.
(A) MTs were marked by transiently expressing TagRFP-MAP-CKL6 (yellow) in N. benthamiana transgenic plants expressing NRIP1-Cerulean that marks stromules (cyan). Confocal micrographs of …
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Figure 1—source data 1
Statistical analysis of the stromule velocity along MT.
A raw data for the average stromule velocity along MT presented in Figure 1D.
- https://doi.org/10.7554/eLife.23625.005
Figure 1—figure supplement 1
Transmission electron microscopy of stromule-to-microtubule interactions.
(A) Stromules were induced by transiently expressing the p50 effector for 42 hr in plants containing the N immune receptor. Serial sections were obtained and an overview montage image showing the …
Figure 2
Microtubules direct stromule movement along the ER.
(A) The endoplasmic reticulum (ER) were marked by transiently expressing SP-Citrine-HDEL (magenta) and MTs were marked by TagRFP-MAP-CKL6 (yellow) in N. benthamiana transgenic lines expressing …
Figure 3
Microtubule disruption lead to stromule retraction and microtubule stabilization increased stromules.
(A) Dynamics of MT depolymerization and stromule retraction in the lower epidermis of NRIP1-Cerulean transgenic N. benthamiana plant leaves after infiltration of mock control (0.2% DMSO) or MT …
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Figure 3—source data 1
Statistics of the stromule frequency under MT disrupting drug treatments.
Raw datapoints of stromule frequency after APM, Oryzalin, or Paclitaxel, presented in Figure 3B and D. Statistical analyses and the original graph generated by Prism7 were also included.
- https://doi.org/10.7554/eLife.23625.012
Figure 4 with 1 supplement
Alterations of MT is correlated with NbGCP4 silencing.
(A) In GFP-TUA6 transgenic N. benthamiana leaves that mark MTs (yellow), NbGCP4 silencing resulted in hyper-parallel and occasional bundling of MT (VIGS-NbGCP4, right) compared to the VIGS vector …
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Figure 4—source data 1
Statistics of the quantitative RT-PCR to validate GCP4 gene silencing.
Statistical analyses and the graph of the quantitative RT-PCR for the NbGCP4 gene silencing presented in Figure 4B were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.016
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Figure 4—source data 2
Statistics of the Azimuthal angles of the MT in VIGS-NbGCP4 plants.
Statistical analyses and the graph of the Azimuthal angles of the MT in the NbGCP4 silenced plants presented in Figure 4C were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.017
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Figure 4—source data 3
Statistics of the curvature of MTs in VIGS-NbGCP4 plants.
Statistical analyses and the graph of the curvature of the MT in the NbGCP4 silenced plants presented in Figure 4E were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.018
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Figure 4—source data 4
Statistics of the SOAX analysis of MTs in VIGS-NbGCP4 plants.
Statistical analyses and the graph of the SOAX analysis of the MT in the NbGCP4 silenced plants presented in Figure 4F were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.019
Figure 4—figure supplement 1
Plant morphology at different days post-silencing of NbGCP4 in NRIP1-Cerulean expressing N. benthamiana plants.
(A) Plants after 4 days of virus induced gene silencing of NbGCP4 (VIGS-NbGCP4) showed no detectable growth or morphological phenotype compared to VIGS vector (VIGS-EV) control plants. (B) Plants …
Figure 5 with 1 supplement
Microtubule stabilization induces stromules constitutively.
(A) Stromules were induced 24 hr after TMV-p50 effector infiltration as described in Caplan et al. (2015) (bottom left) compared to a mock control (top left). Stromules were induced both in mock …
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Figure 5—source data 1
Statistics of the stromule dynamics in VIGS-NbGCP4 plants.
Statistical analyses and the graph of the stromule frequency, length, and velocity in the NbGCP4 silenced plants presented in Figure 5B-D by Prism7 are included.
- https://doi.org/10.7554/eLife.23625.023
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Figure 5—source data 2
Statistics of the MT alteration by TMV-p50 induced immune responses.
Statistical analyses and the graph of the MT distribution presented in Figure 5F-H were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.024
Figure 5—figure supplement 1
Quantitative analysis of stromule length and movements.
(A) Stromule length was manually measured in ImageJ. Consistent with the independent measurement presented in Figure 5C, silencing of NbGCP4 (VIGS-NbGCP4) resulted in a higher percentage of …
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Figure 5—figure supplement 1—source data 1
Statistics of quantitative analysis of stromule length and movement in VIGS-NbGCP4 plants.
The statistics of quantitative analysis of stromule length and movement in VIGS-NbGCP4 plants by Prism7 is included the raw data points, statistical analyses, as well as the original graphs of Figure 5—figure supplement 1A and B.
- https://doi.org/10.7554/eLife.23625.022
Figure 6 with 3 supplements
Stromule are anchored to the actin microfilament network.
(A) AFs were marked by expressing Lifeact-TagRFP (magenta) in N. benthamiana transgenic lines expressing NRIP1-Cerulean that marks stromules (cyan). Stromules were seen interacting with AFs at kink …
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Figure 6—source data 1
Statistics of the quantification of stromules pausing at the AFs.
The raw data points to calculate the percentage of stromules pausing at the AFs during retraction events, presented in Figure 6D.
- https://doi.org/10.7554/eLife.23625.030
Figure 6—figure supplement 1
Chloroplast stromules statically interact with actin microfilaments.
(A) AFs were marked by expressing Lifeact-TagRFP (magenta) in N. benthamiana transgenic lines expressing NRIP1-Cerulean that marks stromules (cyan). Stromules were seen in close proximity to AFs …
Figure 6—figure supplement 2
Characterization of actin microfilaments associated with stromules and the body of chloroplasts.
(A) Stromules were induced by transiently expressing the TMV-p50 effector for 42 hr in plants containing the N NLR immune receptor. A transmission electron micrograph overview montage showing the …
Figure 6—figure supplement 3
Disruption of actin filaments does not affect stromule number.
(A) AFs were marked by transiently expressing Citrine-mTalin (magenta) in N. benthamiana transgenic lines expressing NRIP1-Cerulean that marks stromules (cyan). Leaves were infiltrated with either …
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Figure 6—figure supplement 3—source data 1
Statistics of the stromule frequency after CTD treatment.
Statistical analyses and the stromule frequency after CTD treatment to disrupt actin cytoskeleton presented in Figure 6—figure supplement 3B were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.029
Figure 7 with 1 supplement
Disruption of cytoskeleton change stromule dynamics.
(A) Time lapsed images of stromules 1 hr post-treatment with 1 µM of MT inhibitor oryzalin (ORY) or with 1 µM of actin inhibitor cytochalasin D (CTD) on the leaf of the NRIP1-Cerulean transgenic N. …
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Figure 7—source data 1
Statistics of the stromule dynamics.
All of the statistical analyses and the original graphs of the stromule dynamics presented in Figure 7B-D generated by Prism7 are shown.
- https://doi.org/10.7554/eLife.23625.035
Figure 7—figure supplement 1
Effects of inhibitor treatments on the disruption of cytoskeleton.
N. benthamiana GFP-TUA6 transgenic plants were used to mark microtubule (MT, yellow) and N. benthamiana FABD2-GFP transgenic plants were used for actin filament (AF, magenta). Treatment with 1 µM …
Figure 8 with 2 supplements
Stromule directed chloroplast movement.
(A) Stromules and chloroplast movement events were imaged in NRIP1-Cerulean N. benthamiana transgenic plants. A chloroplast was tracked (red line) in time lapsed images. The direction of movement …
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Figure 8—source data 1
Statistical analyses of the stromule-directed movements.
Statistical analyses and the graph to support the stromule-directed movements presented in Figure 8D and E were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.040
Figure 8—figure supplement 1
Stromule-directed movement.
(A) Stromules and chloroplast movement events were imaged in NRIP1-Cerulean N. benthamiana transgenic plants. (B) The orientation angle of the stromule connected to the chloroplast body (X-axis) and …
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Figure 8—figure supplement 1—source data 1
Data of the stromule-directed movement.
The raw data points of the orientation angle of the stromule and the movement of the chloroplast body presented in Figure 8—figure supplement 1B.
- https://doi.org/10.7554/eLife.23625.038
Figure 8—figure supplement 2
Stromule-directed chloroplast movement during partial actin disruption.
(A) AFs and MTs were marked by transiently expressing Citrine-mTalin (magenta) and TagRFP-MAP-CKL6 (yellow) in N. benthamiana transgenic plants expressing NRIP1-Cerulean that marks stromules (cyan). …
Figure 9 with 1 supplement
Chloroplasts and stromules positioning during perinuclear chloroplast clustering during immune response.
(A) TMV-p50 effector and Lifeact-TagRFP (magenta; AFs) were expressed in transgenic NRIP1-Cerulean N. benthamiana plants that marks stromules (cyan). Time lapse images of stromule retraction toward …
Figure 9—figure supplement 1
Stromule association with perinuclear actin microfilaments.
(A) TMV-p50 effector and Citrine-mTalin (magenta; AFs) were expressed in transgenic NRIP1- Cerulean N. benthamiana plants that marks stromules (Cyan). Stromules interacted with AFs associated with …
Figure 10 with 1 supplement
Perinuclear chloroplast clustering during immune response.
(A) Time course images of perinuclear chloroplasts during TMV-p50 effector induced immune responses compared to mock control. Chloroplasts and stromules marked by NRIP1-Cerulean were pseudocolored …
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Figure 10—source data 1
Statistics of the perinuclear clustering of the chloroplasts in TMV-p50 induced plant immune response and effect of cytoskeleton disrupting drugs.
Statistical analyses and the graph of the perinuclear clustering of the chloroplasts in TMV-p50 induced plant immune response and presented in Figure 10B and D were generated by Prism7.
- https://doi.org/10.7554/eLife.23625.051
Figure 10—figure supplement 1
Perinuclear clustering of chloroplasts in TMV-p50 effector induced immune response.
(A) Frequency map of nuclei associated with different number of chloroplasts in three time points after induction of immunity by TMV-p50 effector. Nucleus number with more than four chloroplasts …
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Figure 10—figure supplement 1—source data 1
Data of the chloroplast clustering to nucleus in TMV-p50 induced immune response.
Nuclei were categorized by the number of chloroplast clustered to the nucleus graphed in Figure 10—figure supplement 1A and B.
- https://doi.org/10.7554/eLife.23625.050
Videos
Video 1
Stromules extend along microtubules.
Stromules were marked by expressing NRIP1(cTP)-TagRFP (Cyan) in GFP-TUA6 (yellow) transgenic N. benthamiana plants. Time-lapse confocal microscopy was used to acquire images every 0.92 s that are …
Video 2
Tracking of stromule tip interactions along MTs.
The first half of the video shows maximum intensity projections of time-lapse confocal microscopy of 120 images taken every 8.1 s and displayed at 8 frames/second. MTs were marked by expressing …
Video 3
Stromule move in channels of ER along MTs.
Video show stromules (cyan) first moving along MTs (yellow) to show dynamic association, then merged with the ER (magenta), and with stromules and ER only to show movement through ER channels. The …
Video 4
Stromules remodeling ER.
Stromules (cyan) move along MTs (yellow) remodeling the ER (magenta). Video was created from three consecutive time series and was used to generate Figure 2B–C.
Video 5
Stromule dynamics are disrupted by APM or Oryzalin MT inhibitor treatment.
Disruption of MT (yellow) by APM (20 µM) resulted in a loss of stromule extensions (middle) compared to the mock treatment control (left). Disruption of MT (yellow) by Oryzalin (300 µM) resulted in …
Video 6
Stromules do not extend along AFs.
Stromules (cyan) interacted with AFs (magenta) at points along the stromule, but stromules did not directly extend along AFs. Sixty images were captured by time-lapse confocal microscopy every 18 s …
Video 7
Stromule retraction to actin anchor points.
Two stromules (cyan) retracted to multiple actin anchor points (arrows) along AFs (magenta). Thirty-seven images were captured by time-lapse confocal microscopy every 18 s and displayed at 12 …
Video 8
Stromule-directed movement.
Chloroplast bodies and stromules were visualized in a NRIP1-CFP transgenic N. benthamiana plants, in combination with chlorophyll autofluorescence. Time-lapse confocal microscopy was used to acquire …
Video 9
Dynamic control of stromules by extension along MTs and anchoring to AFs.
AFs and stromules were marked by transiently expressing Lifeact-TagRFP (magenta) and NRIP1(cTP)-TagBFP (cyan) in N. benthamiana transgenic lines expressing GFP-TUA6 that marks MTs (yellow). Sixteen …
Video 10
Chloroplasts and stromule dynamics following ORY and CTD treatments.
Chloroplast bodies and stromules were visualized in a NRIP1-CFP transgenic N. benthamiana plants, in combination with chlorophyll autofluorescence. Time-lapse confocal microscopy was used to acquire …
Video 11
Stromule directed movement in Cytochalasin-D-treated cell.
Stromules (cyan) from two chloroplasts are disrupted after Cytochalasin D treatment that fragmented AFs (magenta). Stromules re-extended along MTs (yellow) resulting in the movement of chloroplasts. …
Video 12
Stromule retraction resulted in the movement of a chloroplast to a nucleus.
A stromule (cyan) attached to an AF (magenta) pulls the chloroplast body to the nucleus by retracting. 100 maximum intensity projections of time-lapse z-stacks were taken every 38 s and displayed at …
Tables
Key resources table
| Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|
| NRIP1 | PMID: 18267075; 26120031 | ||
| GCP4 | This paper | ||
| p50 | PMID: 18267075; 26120031 | ||
| GV2260 | PMID: 4022773 | ||
| GV3101 | https://doi.org/10.1007/BF00331014 | ||
| NRIP1-Cerulean transgenic plant | PMID: 18267075; 26120031 | ||
| GFP-TUA6 transgenic plant | PMID:12084822 | ||
| FABD2-GFP transgenic plant | PMID:12084822 | ||
| NRIP-Cerulean; N double transgenic plant | PMID: 18267075; 26120031 | ||
| TRV1 | PMID: 14501071 | ||
| TRV2-EV | PMID: 14501071 | ||
| Nicotiana benthamiana | Taxonomy ID: 4100 | ||
| mTalin-Citrine | This paper | ask construct named ‘SPKD2681’ | |
| Lifeact-TagRFP | This paper | ask construct named ‘SPKD2209’ | |
| TagRFP-MAP-CKL6 | This paper | ask construct named ‘SPKD2386’ | |
| NRIP1(cTP)-TagBFP | This paper | ask construct named ‘SPKD3168’ | |
| TRV-NbGCP4 | This paper | ask construct named ‘SPKD3111’ | |
| Citrine-p50-U1 | This paper | ask construct named ‘SPKD1939’ | |
| Citrine | This paper | ask construct named ‘SPKD914’ | |
| p50-2xHA | PMID: 18267075; 26120031 | ||
| NLS-mCherry | PMID: 28619883 | ||
| Cytochalasin D | Sigma-Aldrich, St. Louis, MO | C8273 | |
| Aminoprophos-methyl (APM) | Sigma-Aldrich, St. Louis, MO | 03992 | |
| Oryzalin | Sigma-Aldrich, St. Louis, MO | 36182 | |
| Paclitaxel-BODIPY | Thermofisher Scientific, Waltham, MA | P7501 | |
| DMSO | Sigma-Aldrich, St. Louis, MO | D8418 | |
| PRISM7 | GraphPad, La Jolla, CA | ||
| Stromule detection and tracking algorithm | http://sigport.org/1807 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.23625.052
