Chemical screening pipeline for identification of specific plant autophagy modulators

Abstract

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 ...