Article Figures & Data
Figures
- Figure 1.
R3a Relocalizes to Vesicles in the Presence of Recognized Effectors.
(A) Confocal laser scanning microscopy following transient expression (by agroinfiltration) in N. benthamiana of YFP-R3a alone or transiently coexpressed with either AVR3aEM, AVR3aKI, AVR2, Pex147-2, or Pex147-3 as indicated in the panels at 2 d after inoculation. Experiments were repeated at least five times. Bar = 25 µm.
(B) Immunoblot probed with α-GFP following transient expression of YFP-R3a alone or coexpressed with AVR3aEM or AVR3aKI in N. benthamiana at 2 d after inoculation. Protein sizes are indicated (in kilodaltons), and protein loading is shown by Ponceau stain (PS).
- Figure 2.
Relocalization of R3a Occurs before Development of HR Symptoms.
(A) Confocal laser scanning microscopy following transient expression (by agroinfiltration) in N. benthamiana of YFP-R3a alone or in the presence of conditionally coexpressed Avr3aEM and Avr3aKI at the indicated time points after dexamethasone treatment at 2 d after inoculation. Bar = 20 µm.
(B) Transient expression of R3a alone or in the presence of conditionally coexpressed Avr3aEM and Avr3aKI by agroinfiltration in N. benthamiana. Leaf photos were taken at indicated time points after dexamethasone treatment and are representative of three independent assays. Circles indicate the infiltrated area on the leaf panel.
(C) Close-up of leaf areas transiently expressing R3a with conditionally coexpressed Avr3aKI by agroinfiltration in N. benthamiana. Photos were taken at indicated time points after dexamethasone treatment.
- Figure 3.
In the Presence of Recognized Effectors, R3a Relocalizes to Endosomal Compartments.
Colocalization studies using confocal laser scanning microscopy following transient coexpression (by agroinfiltration) in N. benthamiana of GFP-R3a or mRFP-R3a with 35S-driven AVR3aKI and different subcellular markers: Ara6-mRFP, mRFP-Ara7, PS1-CFP (blue PVC marker), ST-YFP (yellow Golgi-marker; same as in Supplemental Figure 2 online). Colocalization of GFP-R3a with FM4-64 (coexpressed with 35S:AVR3aKI) was examined 20 min after FM4-64 infiltration. Arrow indicates vesicle stained with FM4-64 but not GFP-R3a as described in the text. Left picture shows FP-R3a fluorescence, right picture shows marker fluorescence, and middle picture shows overlay of left and right pictures, all taken at 2 d after inoculation. Images showing colocalization of YFP-R3a with PS1-CFP, following coexpression with dex:AVR3aKI, were taken 2 h after dexamethasone treatment. Bars = 10 µm.
- Figure 4.
Avr3aKI, but Not Avr3aEM, Relocalizes to Late Endosomes in the Presence of Untagged R3a Prior to HR Development.
(A) Confocal laser scanning microscopy following transient coexpression by agroinfiltration in N. benthamiana of GFP-AVR3aKI (left picture) and mRFP-R3a (right picture), with overlay (middle picture) indicating colocalization of GFP and mRFP fluorescence at 2 d after inoculation.
(B) GFP-Avr3aKI or GFP-Avr3aEM (left picture, as indicated), each coexpressed with PVC marker PS1-CFP (right picture) and untagged R3a, with overlay (middle picture), indicating colocalization of GFP and CFP fluorescence only in the case of GFP-Avr3aKI.
(C) GFP-Avr3aKI (left picture) coexpressed with untagged R3a. Localization of FM4-64 (right picture) was examined 20 min after its infiltration. Overlay (middle picture) indicates colocalization of GFP and FM4-64 fluorescence. For experiments in (B) and (C), agroinfiltration to express untagged R3a was performed 24 h after agroinfiltration to express GFP-Avr3a (and PS1-CFP, as indicated). Images in (B) and (C) were taken 2 d after inoculation of GFP-AVR3a (1 d after inoculation for R3a). Bars = 10 µm.
(D) Transient expression of untagged R3a and GFP-Avr3aKI alone or coexpressed (agroinfiltration to express R3a was 24 h after agroinfiltration to express GFP-Avr3a). Photos of HRs were taken at indicated time points (h p.i., hours post inoculation) and are representative of multiple independent assays. Circles indicate the infiltrated area on the leaf panel.
- Figure 5.
BiFC Indicates Close Proximity of R3a and AVR3aKI in Planta.
(A) and (C) Confocal laser scanning microscopy following transient coexpression in N. benthamiana by agroinfiltration of split-YFP constructs YN-R3a with either YC-AVR3aEM (left picture) or YC-AVR3aKI (right picture), as indicated, at 2 d after inoculation (A) and split-YFP constructs YN-R3a and YC-Avr3aKI, coexpressed with the PVC marker PS1-CFP, at 2 d after inoculation (C). Overlay (middle picture) indicates colocalization of YFP fluorescence (left picture) and CFP fluorescence (right picture). Bars = 20 µm.
(B) Immunoblot probed with α-cMyc or α-HA following transient expression of split-YFP constructs shown in (A) in N. benthamiana at 3 d after inoculation. Protein sizes are indicated (in kilodaltons) and protein loading is shown by Ponceau S (PS). Experiments in (A) and (C) were repeated on four occasions.
- Figure 6.
Wortmannin and BFA Attenuate R3a Relocalization.
(A) Confocal laser scanning microscopy following transient coexpression in N. benthamiana by agroinfiltration of YFP-R3a with AVR3aKI in the absence (-wortmannin, -BFA, left pictures) and 30 min after infiltration of 33 µM Wortmannin or 20 min after infiltration of 20 μg/mL BFA (right picture). Bar = 25 (top images) and 20 (bottom images) µm. Pictures were taken 2 d after agroinfiltration, and experiments were repeated three times.
(B) Immunoblot probed with α-GFP following transient coexpression of YFP-R3a with AVR3aKI (left picture) and transient coexpression of GFP-Avr3aKI with mRFP-R3a (right picture) in N. benthamiana by agroinfiltration at 2 d after inoculation to show stability of R3a and Avr3a after application of 20 µg/mL BFA and 30 µM wortmannin for 2 h. Protein sizes are indicated (in kilodaltons), and protein loading is shown by Ponceau S (PS).
- Figure 7.
The Autoactive Mutant R3a(D501V) Is Cytoplasmic and Triggers HR in the Absence of Effectors.
(A) The MHD mutant of R3a used in this work. Wild-type sequence (top) and mutant sequence (bottom) is shown.
(B) Transient expression of R3a and R3a(D501V) alone and coexpression of R3a and AVR3aKI by agroinfiltration in N. benthamiana. Photo of HRs was taken 4 d after inoculation and is representative from multiple assays. Circles indicate the infiltrated area on the leaf panel.
(C) Immunoblot probed with α-GFP following transient expression of YFP-R3a(D501V) in N. benthamiana at 2 d after inoculation. Protein sizes are indicated (in kilodaltons), and protein loading is shown by Ponceau S (PS). A leaf sample constitutively expressing GFP was used as a positive control.
(D) Confocal laser scanning microscopy following transient expression in N. benthamiana of YFP-R3a alone (top left picture), YFP-R3a coexpressed with AVR3aKI (top right picture), YFP-R3a(D501V) alone (bottom left picture), and YFP-R3a(D501V) coexpressed with AVR3aKI (bottom right picture) at 2 d after inoculation. Bar = 20 µm.
- Figure 8.
BFA and Wortmannin Significantly Attenuate HR Development Triggered by R3a in the Presence of AVR3aKI in Contrast with R3a(D501V)- and Rx1-Triggered HR.
(A) and (B) Transient coexpression of R3a with AVR3aKI (left picture) and Rx1(Rx) with PVX-CP (right picture) and expression of R3a(D501V) (middle picture) by agroinfiltration in N. benthamiana (red circles) was followed 24 h later [48 h later for R3a(D501V)-expressing leaves] by infiltration of 20 µg/mL BFA (A) and 33 µM wortmannin (B) (areas infiltrated by inhibitors indicated as blue circles). Photos of HRs were taken 48 h [72 h for R3a(D501V)-expressing leaves] after agroinfiltration.
(C) Graph shows the percentage of inhibitor (BFA and wortmannin) infiltration sites developing a clear HR at 2 d (for R3a and Rx1) and 3 d [for R3a(D501V)] after agroinfiltration. Experiments were repeated at least three times, each with no less than four leaves from four plants. Error bars indicate ± se.
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