Abstract
Extracellular vesicles (EVs) produced by Arabidopsis (Arabidopsis thaliana) plants are highly heterogeneous in protein content. To understand the origins of plant EV heterogeneity, we used an unbiased proximity labeling approach to identify proteins and pathways involved in the secretion of distinct EV subpopulations. Proximity labeling, co-immunoprecipitation, and fluorescence microscopy in Nicotiana benthamiana all indicated a general role in EV secretion for EXO70 proteins (a subunit of the exocyst complex) and the immune-related protein RPM1-INTERACTING PROTEIN4 (RIN4). To confirm these hypotheses, we assessed the impact of mutations in various EXO70 family genes and in RIN4 on EV release, as well as mutations in additional genes known to regulate endomembrane trafficking and secretion. Mutation of EXO70E1, EXO70E2 or STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1; a GTP-exchange factor for RabE GTPases) reduced secretion of EVs marked by TETRASPANIN8 (TET8), PENETRATION1 (PEN1), and PATELLIN1 (PATL1), indicating that these proteins are generally required for EV secretion. In contrast, mutation of RIN4 reduced levels of TET8+ and PEN1+ EVs, but not PATL + EVs. Mutation of the small GTPase gene RABA2a specifically affected PEN1+ EV secretion, while mutations in AUTOPHAGY PROTEIN5 (ATG5) and VAMP-ASSOCIATED PROTEIN27 (VAP27) specifically affected TET8+ EV secretion. Lastly, we found that exo70 family mutants are more susceptible to infection with the fungal pathogen Colletotrichum higginsianum, underlining the importance of secretion for plant immunity. Together, our results unravel some of the complex mechanisms that give rise to EV subpopulations in plants.
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Claims 8
Claim · model-written summary
Proximity labeling, co-immunoprecipitation and fluorescence microscopy in Nicotiana benthamiana indicated a general role of EXO70 proteins in EV secretion.
- Taxon
- Nicotiana benthamiana
Nicotiana benthamianae1 - Gene
- EXO70 proteinse1
- Outcome
- EV secretione1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“fluorescence microscopy in Nicotiana benthamiana all indicated a general role in EV secretion for EXO70 proteins (a subunit of the exocyst complex) and the immune-related protein RPM1-INTERACTING PROTEIN4 (RIN4)”
Claim · model-written summary
Proximity labeling, co-immunoprecipitation and fluorescence microscopy in Nicotiana benthamiana indicated a general role of RIN4 in EV secretion.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Gene
- RPM1-INTERACTING PROTEIN4 (RIN4)e1
- Outcome
- EV secretione1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“fluorescence microscopy in Nicotiana benthamiana all indicated a general role in EV secretion for EXO70 proteins (a subunit of the exocyst complex) and the immune-related protein RPM1-INTERACTING PROTEIN4 (RIN4)”
Claim · model-written summary
Mutation of EXO70E1, EXO70E2 or SCD1 reduced secretion of TET8-, PEN1- and PATL1-marked EVs in Arabidopsis.
- Taxon
- Arabidopsis
Arabidopsise0 - Treatment
- Mutation of EXO70E1, EXO70E2 or STOMATAL CYTOKINESIS DEFECTIVE1e1
- Gene
- EXO70E1, EXO70E2 or STOMATAL CYTOKINESIS DEFECTIVE1e1
- Outcome
- secretion of EVse1
- Direction
- reducede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“Mutation of EXO70E1, EXO70E2 or STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1; a GTP-exchange factor for RabE GTPases) reduced secretion of EVs marked by TETRASPANIN8 (TET8), PENETRATION1 (PEN1), and PATELLIN1 (PATL1)”
Claim · model-written summary
Mutation of RIN4 reduced levels of TET8+ and PEN1+ EVs in Arabidopsis.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- mutation of RIN4e1
- Gene
- RIN4e1
- Outcome
- levels of TET8+ and PEN1+ EVse1
- Direction
- reducede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“mutation of RIN4 reduced levels of TET8+ and PEN1+ EVs”
Claim · model-written summary
Mutation of RIN4 did not reduce levels of PATL+ EVs in Arabidopsis.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- mutation of RIN4e1
- Gene
- RIN4e1
- Outcome
- PATL + EVse1
- Direction
- no_changeinferrede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“mutation of RIN4 reduced levels of TET8+ and PEN1+ EVs, but not PATL + EVs”
Claim · model-written summary
Mutation of the small GTPase gene RABA2a specifically affected PEN1+ EV secretion in Arabidopsis.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- Mutation of the small GTPase gene RABA2ae1
- Gene
- RABA2ae1
- Outcome
- PEN1+ EV secretione1
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“Mutation of the small GTPase gene RABA2a specifically affected PEN1+ EV secretion”
Claim · model-written summary
Mutations in ATG5 and VAP27 specifically affected TET8+ EV secretion in Arabidopsis.
- Taxon
- ArabidopsisArabidopsise0
- Treatment
- mutations in AUTOPHAGY PROTEIN5 (ATG5) and VAMP-ASSOCIATED PROTEIN27 (VAP27)e1
- Gene
- AUTOPHAGY PROTEIN5 (ATG5) and VAMP-ASSOCIATED PROTEIN27 (VAP27)e1
- Outcome
- TET8+ EV secretione1
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“mutations in AUTOPHAGY PROTEIN5 (ATG5) and VAMP-ASSOCIATED PROTEIN27 (VAP27) specifically affected TET8+ EV secretion”
Claim · model-written summary
exo70 family mutants were more susceptible to infection with the fungal pathogen Colletotrichum higginsianum.
- Taxon
- ArabidopsisArabidopsise0
- Material
- exo70 family mutantse1
- Interactor
- Colletotrichum higginsianumColletotrichum higginsianume1pathogen
- Outcome
- susceptible to infectione1
- Direction
- moree1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Arabidopsis (Arabidopsis thaliana)”
e1“exo70 family mutants are more susceptible to infection with the fungal pathogen Colletotrichum higginsianum”