An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor
- The Sainsbury Laboratory, United Kingdom
- John Innes Centre, United Kingdom
- Imperial College London, United Kingdom
Figures
Figure 1 with 1 supplement
PexRD54 binds to ATG8CL via a C-terminal ATG8 interacting motif (AIM).
(A) Domain organization of PexRD54. PexRD54 is a canonical RXLR effector with five WY folds (Win et al., 2012). The amino acid sequences of candidate AIMs are highlighted in yellow color and …
Figure 1—figure supplement 1
A synthetic peptide composed of the last ten amino acids of PexRD54 is sufficient for binding to ATG8CL.
(A) A synthetic peptide (KPLDFDWEIV) containing the AIM of PexRD54 binds ATG8CL as documented by ITC. (B) A mutant AIM peptide (KPLDFDAEIA) was used as negative control and did not bind ATG8CL. Heat …
Figure 2 with 2 supplements
PexRD54 has higher binding affinity to ATG8CL than ATG8IL.
(A) RFP:PexRD54, RFP:AVRblb2 or RFP:EV were transiently co-expressed with GFP:ATG8CL, GFP:ATG8IL or GFP:EV in N. benthamiana leaves and proteins were extracted two days after infiltration and used …
Figure 2—figure supplement 1
Amino acid sequence alignment of ATG8 proteins from Arabidopsis thaliana (At), Solanum tuberosum (St), Solanum lycopersicum (Sl) and Nicotiana benthamiana (Nb).
Sequences were aligned using ClustalW and visualized using Boxshade v3.21. Identical amino acids are highlighted in black and amino acids with similar biochemical properties are shown in grey. Red …
Figure 2—figure supplement 2
Phylogenetic analysis of ATG8 proteins.
A neighbor-joining tree was generated with ATG8 sequences obtained from genomes of A. thaliana, S. tuberosum, S. lycopersium, and N. benthamiana, using MEGA5 with a 1000 bootstrap value. ATG8CL and …
Figure 3 with 7 supplements
PexRD54 localizes to ATG8CL labelled autophagosomes.
Transient co-expression of GFP:ATG8CL with (A) RFP:PexRD54, (B) RFP:PexRD54AIM2 and (C) RFP control (RFP:EV) in N. benthamiana leaves. Confocal micrographs show single optical sections of …
Figure 3—figure supplement 1
PexRD54 localizes to vesicle like structures.
GFP:PexRD54 or RFP:PexRD54 were transiently expressed in N. benthamiana and examined by confocal laser scanning microscopy 3 days post infiltration. Confocal micrographs show single optical sections …
Figure 3—figure supplement 2
PexRD54 localizes to high electron dense structures.
(A) Transmission electron microscopy (TEM) imaging of PexRD54. N. benthamiana cells transiently expressing GFP:PexRD54 or GFP:EV were collected 3 days post infiltration and probed with anti-GFP …
Figure 3—figure supplement 3
PexRD54 labelled high electron dense structures that are not peroxisomes.
Serial sections of N. benthamiana leaves transiently expressing GFP:PexRD54 were collected 3 days post infiltration and probed with anti-GFP and anti-catalase antibodies conjugated to gold …
Figure 3—figure supplement 4
PexRD54 labelled puncta rarely include GFP:ATGIL signal.
GFP:ATG8IL was transiently co-expressed with RFP:PexRD54 or RFP:EV in N. benthamiana leaves and examined by confocal laser scanning microscopy 3 days post infiltration. Maximum intensity projection …
Figure 3—figure supplement 5
Inhibition of autophagy hinders PexRD54 vesicular distribution.
N. benthamiana leaves transiently expressing GFP:ATG8CL/RFP:PexRD54, GFP:PexRD54/RFP:ATG8CL or trans-golgi network marker YFP:VTI12 were infiltrated with autophagy inhibitor 3-methyl adenine (3-MA) …
Figure 3—figure supplement 6
Conservation of terminal glycine residue in ATG8 proteins from various species.
Amino acid sequence alignment of ATG8 variants from different species that belong to different kingdoms shows conservation of terminal glycine residue (highlighted with an orange arrow). Residues …
Figure 3—figure supplement 7
ATG8CL terminal glycine deletion mutant prevents formation of PexRD54 labelled puncta in a dominant negative manner.
(A) A glycine to alanine mutation in the conserved terminal glycine residue of GFP:ATG8CL does not prevent binding to RFP:PexRD54. GFP:ATG8CL or GFP:ATG8CLΔ were transiently co-expressed with …
Figure 4
PexRD54 increases the number of ATG8CL autophagosomes.
(A) Co-expression of RFP:PexRD54, but not RFP:PexRD54AIM2 or RFP:EV significantly enhanced the number of GFP:ATG8CL-labelled autophagosomes in N. benthamiana. Bar charts display the number of …
Figure 5 with 1 supplement
PexRD54 does not block autophagic flux.
(A–B) ConcanamycinA treatment increases the number of autophagosomes in PexRD54 expressing samples.RFP:ATG8CL was transienly coexpressed with PexRD54, PexRD54AIM2 and empty vector controls in N. …
Figure 5—figure supplement 1
GFP:ATG8CL protein levels increase in N. benthamiana during P. infestans infection.
N. benthamiana leaves transiently expressing GFP:ATG8CL or GFP:EV were sprayed with P. infestans zoospore solution or water control (mock treatment). Protein levels of GFP:ATG8CL or GFP were …
Figure 6 with 6 supplements
PexRD54 is a competitive antagonist of the plant selective autophagy cargo receptor Joka2.
(A) Domain organization of Joka2. (B) PexRD54 reduces binding of Joka2 to ATG8CL in a dose-dependent manner. GFP:ATG8CL (OD=0.2) and Joka2:RFP (OD=0.2) were transiently co-expressed with varying …
Figure 6—figure supplement 1
Joka2 has a functional ATG8 interacting motif (AIM).
Mutation of the predicted AIM in Joka2 (819-WDPI-822) to ADPA resulted in loss of ATG8CL binding. GFP:EV and GFP:ATG8CL were co-expressed with Joka2:RFP and Joka2AIM:RFP in N. benthamiana and …
Figure 6—figure supplement 2
Joka2 has higher binding affinity to ATG8CL than ATG8IL.
Joka2:RFP was transiently co-expressed with GFP:ATG8CL, GFP:ATG8IL or GFP:EV in N. benthamiana and proteins were extracted two days after infiltration and used in immunoprecipitation experiments …
Figure 6—figure supplement 3
Joka2 increases the number of ATG8CL labelled autophagasomes.
(A) Joka2:RFP partially colocalizes with GFP:ATG8CL. Joka2:RFP and GFP:ATG8CL were transiently co-expressed in N. benthamiana leaves and examined by confocal laser scanning microscopy 3 days post …
Figure 6—figure supplement 4
Joka2 increases ATG8CL protein levels.
GFP:ATG8CL was transiently co-expressed with RFP:GUS, Joka2:RFP or Joka2AIM:RFP in N. benthamiana leaves and protein levels in total extracts were determined 2 and 3 days after infiltration, using …
Figure 6—figure supplement 5
PexRD54 outcompetes Joka2 for ATG8CL binding.
Joka2:RFP and GFP:ATG8CL were transiently co-expressed with HA:EV, HA:PexRD54 and HA:PexRD54AIM2 in N. benthamiana leaves and proteins were extracted two days after infiltration and used in …
Figure 6—figure supplement 6
PexRD54 AIM has a higher affinity to ATG8CL than Joka2 AIM.
GFP:ATG8CL and Joka2:RFP were transiently co-expressed with HA:PexRD54, HA:PexRD54AIM2, HA:PexRD54J2AIM1, HA:PexRD54 J2AIM2 in N. benthamiana and proteins were extracted two days after infiltration …
Figure 7 with 1 supplement
PexRD54 counteracts the enhanced resistance conferred by Joka2.
(A) Overexpression of Joka2:RFP limits P. infestans colonization. Halves of N. benthamiana leaves expressing RFP:EV, Joka2:RFP and Joka2AIM:RFP were infected with P. infestans and pathogen growth …
Figure 7—figure supplement 1
Silencing of Joka2 enhances susceptibility to P. infestans.
(A) Cartoon showing Joka2 sites targeted for VIGs. (B) TRV:Joka2-1, TRV:Joka2-2 and TRV:GFP control plants were inoculated with P. infestans 88,069 and pathogen growth was determined by lesion areas …
Figure 8
PexRD54 outcompetes the autophagy cargo receptor Joka2 and enhances virulence.
https://doi.org/10.7554/eLife.10856.032Videos
Video 1
PexRD54 localizes to mobile endomembrane compartments.
GFP:PexRD54 was transiently expressed in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 3)
Video 2
PexRD54 and ATG8CL colocalize at mobile endomembrane compartments.
RFP:PexRD54 was transiently co-expressed with GFP:ATG8CL in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 3)
Video 3
RFP:PexRD54 and GFP:ATG8CL colocalize at mobile ring shaped clusters.
RFP:PexRD54 was transiently co-expressed with GFP:ATG8CL in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 4)
Video 4
RFP:ATG8CL and Joka2:GFP colocalize at mobile endomembrane compartments.
RFP:ATG8CL was transiently coexpressed with Joka2:GFP in N. benthamiana leaves and examined by confocal laser scanning microscopy, 3 days post infiltration. (Related to Figure 6)
Additional files
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Supplementary file 1
Plant proteins that associate with PexRD54 as identified by mass spectrometry after immunoprecipitation (IP).
FLAG:PexRD54 was transiently expressed in N. benthamiana leaves and proteins were extracted two days after infiltration and used in IP experiments. Unique spectral counts are given for each control and PexRD54 sample. Proteins related to vesicle trafficking and autophagy are highlighted in yellow. ATG8CL is highlighted in blue and found in several replicates including the stringent IP experiment.
- https://doi.org/10.7554/eLife.10856.033
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Supplementary file 2
Unique peptides obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of PexRD54 immunoprecipitation experiments suggest specific association with ATG8CL.
Although ATG8 protein family is highly conserved, and there are eight ATG8 variants in N. benthamiana, unique peptides obtained from LC-MS/MS analysis enabled specific identification of ATG8CL variant (Nb_S00003316g0005) from N. benthamiana. ATG8CL variant of N. benthamiana has identical amino acid sequence with the potato ATG8CL variant that is used for other experiments.
- https://doi.org/10.7554/eLife.10856.034
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Supplementary file 3
Primers used in this study.
- https://doi.org/10.7554/eLife.10856.035
