Exocyst and autophagy-related membrane trafficking in plants

Pečenková, Tamara; Marković, Vedrana; Sabol, Peter; Kulich, Ivan; Žárský, Viktor

doi:10.1093/jxb/erx363

Cited by 47 publications

(46 citation statements)

References 109 publications

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“…Autophagy is integrated tightly with endocytic and exocytic pathways and the trafficking routes to the vacuole (Deretic et al, 2012;Spitzer et al, 2015;Pečenková et al, 2017). Thus, it is probable that putative modulators of autophagy identified in the previous phases of the screen influence the endomembrane system and thus activate or inhibit autophagy indirectly and have significant off-target effects, as was recently shown for some characterized compounds (Wu et al, 2010; Mauthe et al, 2018).…”

Section: Phase 4: Specificity Checkmentioning

confidence: 86%

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators

et al. 2019

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Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

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Section: Phase 4: Specificity Checkmentioning

confidence: 86%

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators

et al. 2019

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“…EXO70s are subunits of the octomeric exocyst complex, which is involved in exocytosis and other cellular trafficking processes. Recent studies have shown that EXO70 paralogs could also play roles in autophagy 17, 50, 51 . EXO70D3 interacted with ARR5 D87E , but not with a wild-type ARR5 or ARR5 D87A bait in a yeast two-hybrid assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with this, EXO70B1 colocalizes with ATG8 proteins in autophagosomes and disruption of EXO70B1 resulted in fewer vacuolar autophagic vesicles 17 . Multiple other EXO70 isoforms also contain AIM domains 18, 19 , suggesting that they may also act as receptors for the autophagic regulation of various cellular components.…”

Section: Introductionmentioning

confidence: 99%

EXO70D isoforms mediate selective autophagic degradation of Type-A ARR proteins to regulate cytokinin sensitivity

Acheampong

Shanks

Chang

et al. 2020

Preprint

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19The phytohormone cytokinin influences many aspects of plant growth and development, several of 20 which also involve the cellular process of autophagy, including leaf senescence, nutrient re-21 mobilization, and developmental transitions. The Arabidopsis type-A Response Regulators (type-A 22 ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to 23 cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, 24 demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A 25 ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED 26 (ATG)5-dependent manner. EXO70D family members interacted with Type-A ARR proteins, likely 27 in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction with 28 the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants 29 compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR 30 proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 31 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and 32 cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act 33 as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, 34 demonstrating that cytokinin signaling can be modulated by selective autophagy. 35 36 37Autophagy is a major catabolic pathway that maintains cellular homeostasis in response to 38 intrinsic developmental changes and environmental cues. It mediates the degradation of protein 39 complexes, misfolded and aggregated proteins, and damaged organelles by targeting proteins to 40 proteases localized in the vacuole or lysosome, with a subsequent retro-transport of cellular building 41 blocks back into the cytosol 1-3 . There are three main types of autophagy: macro-, micro-and 42 chaperon-mediated autophagy 1,4,5 . Macro-autophagy (hereafter referred to simply as autophagy) is 43 mediated by conserved AUTOPHAGY-RELATED GENEs (ATGs) that coordinate the de novo 44 biogenesis of the autophagy organelle, the autophagosome 6-8 . Autophagosomes are double 45 membrane vesicles that sequester various cargoes and ultimately deliver them to the lytic vacuole, 46 resulting in their degradation and subsequent recycling. Although basal autophagy occurs in cells 47 under steady-state conditions 9,10 , it is enhanced in response to biotic and abiotic stresses 11-13 and can 48 result in selective or bulk (non-selective) degradation of proteins 4 . Typically, bulk autophagy 49 randomly degrades cytosolic content, while selective autophagy requires unique receptors to target 50 specific cargos for degradation 14,15 . These receptors contain the unique ATG8-INTERACTING 51 MOTIFs (AIM) or LIR-INTERACTING MOTIFs that allow them to interact with the ATG proteins 52 on the autophagosomal membrane 16 . They also...

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“…Autophagy is tightly integrated with endo- and exocytic pathways and the trafficking routes to the vacuole 24–26 . Thus, it is probable that putative modulators of autophagy identified in the previous phases of the screen might influence the endomembrane system and thus activate or inhibit autophagy indirectly and have significant off-target effects, as it has been recently shown for some of the known compounds 9, 10 .…”

Section: Resultsmentioning

confidence: 99%

“…While assays of the first three phases are focused on assessing the drug effect on autophagy activity, the fourth phase puts the drug activity into a broader context. Due to overwhelming complexity of the network, it is frequently overlooked that autophagy is an integral part of the endomembrane trafficking system 26, 29, 30 . The assays in the fourth phase help to identify treatment conditions under which the candidate drugs significantly influence autophagy activity while not causing detectable aberrations in other components of the trafficking machinery.…”

Section: Discussionmentioning

confidence: 99%

Chemical screening pipeline for identification of specific plant autophagy modulators

Dauphinee

Cardoso

Dalman

et al. 2019

Preprint

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16Autophagy is a major catabolic process in eukaryotes and is implicated in responses to biotic 17 and abiotic stresses, programs of aging and cell death. Autophagy is known to regulate 18 agriculturally important traits such as plant fitness, longevity, biomass and seed yield. 19 However, despite its obvious significance, there is still a shortage of reliable tools modulating 20 plant autophagy. Here we describe the first robust pipeline for identification of specific plant 21 autophagy-modulating compounds. Our novel screening protocol comprises four phases: (i) 22 high-throughput screening of chemical compounds in cell cultures of Nicotiana tabacum; (ii) 23 confirmation of the identified hits in planta using Arabidopsis thaliana; (iii) further 24 characterization of the effect using conventional molecular biology methods; (iv) verification 25 of specificity of the effect to autophagy in planta. The methods detailed here streamline 26 identification of specific plant autophagy modulators and greatly aid in unravelling the 27 molecular mechanisms of plant autophagy. 28 Main 29Autophagy is a key regulator of eukaryotic cellular homeostasis and plays an essential role in 30 abiotic and biotic stress responses, fecundity, aging and cell death 1-5 . The autophagy pathway 31 comprises selective or bulk sequestration of cytosolic contents into double-membraned vesicles 32 known as autophagosomes and their delivery to the lytic compartment, where autophagy 33 activity (or autophagic flux) completes with the degradation of the delivered cargo 6 . Autophagy 34 in eukaryotes is coordinated by evolutionary conserved autophagy-related (ATG) proteins that 35 orchestrate sequestration of the cargo, formation and trafficking of the autophagosomes and 36 cargo degradation. Among these proteins, ATG8 is remarkable in the sense that it is 37 incorporated into the membranes of forming autophagosomes, trafficked to the lytic 38 compartment and degraded together with the cargo, making it an ideal tool for autophagy 39 activity read-out 7 . 40 The importance of autophagy for plant stress tolerance in general and crop performance in 41 particular is of critical interest in plant biology, requiring continuous development of 42 increasingly sophisticated tools for its investigation 1,8 . The forward and reverse genetic 43 approaches such as knockouts, knockdowns and overexpression of ATG genes, although being 44 effective tools, have certain limitations 1,7 . The chemical modulation of autophagy presents an 45 alternative option and has also been instrumental for our current understanding of the 46 3 underlying mechanisms controlling autophagy 7 . However, an increasing number of studies 47 describe significant off-target effects of commonly used autophagy-modulating drugs. For 48 example, 3-methyladenine (3-MA) that is typically used as an inhibitor of autophagy 7 , can also 49 increase autophagic flux after prolonged treatments due to its transient suppression of class III 50 phosphoinositide 3-kinases (PI3Ks) 9 . Another ...

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Exocyst and autophagy-related membrane trafficking in plants

Cited by 47 publications

References 109 publications

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators

EXO70D isoforms mediate selective autophagic degradation of Type-A ARR proteins to regulate cytokinin sensitivity

Chemical screening pipeline for identification of specific plant autophagy modulators

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