Techniques to Study Autophagy in Plants

Autophagy (or self eating), a cellular recycling mechanism, became the center of interest and subject of intensive research in recent years. Development of new molecular techniques allowed the study of this biological phenomenon in various model organisms ranging from yeast to plants and mammals. Accumulating data provide evidence that autophagy is involved in a spectrum of biological mechanisms including plant growth, development, response to stress, and defense against pathogens. In this review, we briefly summarize general and plant-related autophagy studies, and explain techniques commonly used to study autophagy. We also try to extrapolate how autophagy techniques used in other organisms may be adapted to plant studies

RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates and damaged organelles under cellular stress conditions. Following sequestration in double or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (Receptor Activated C-Kinase 1, GNB2L1), as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mTOR blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(®) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field