In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

[Abstract unavailable

Plants sense potential microbial invaders by using patternrecognition receptors to recognize pathogen-associated molecular patterns (PAMPs) 1 . In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref.2) and elongation factor Tu receptor (EFR) (ref.3) act as pattern-recognition receptors for the bacterial PAMPs flagellin 4 and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucinerich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellintriggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.PAMPs have key roles as activators of the innate immune response in animals 8 and, analogously, as 'general elicitors' of defence responses in plants [1][2][3][4][5][9][10][11] . We have previously characterized FLS2 and EFR as the pattern-recognition receptors (PRRs) for flagellin (represented by a 22-amino-acid peptide, flg22) and for EF-Tu (represented by the peptides elf18 and elf26, which correspond to its amino terminus), respectively 2-5,10 . Flagellin and EF-Tu rapidly induce a common set of Arabidopsis genes for leucine-rich repeat receptor-like kinases (LRR-RLKs), including FLS2 and EFR themselves 3,11 . This led to the assumption that some of these PAMPinduced LRR-RLKs might encode additional components of PAMP perception or signalling. Using a reverse genetic approach, we tested a collection of insertional mutants in these LRR-RLKs (previously used for identification of the EFR gene 3 ) for responsiveness to flg22 and found that two mutants with insertions in the LRR-RLK gene At4g33430 have reduced sensitivity to flg22 in seedling growth assays (Fig. 1a,b). In more than 10 repetitions of seedling growth assays with these mutants, we always observed a clear reduction (but never a complete loss) of sensitivity to flg22 and flg22-related peptides. By contrast, the mutants seemed to be as sensitive as the wild type to treatment with elf18 in more than five seedling growth assays (Fig. 1b and data not shown).

Innate immunity constitutes the first line of defense against attempted microbial invasion, and it is a well-described phenomenon in vertebrates and insects. Recent pioneering work has revealed striking similarities between the molecular organization of animal and plant systems for nonself recognition and anti-microbial defense. Like animals, plants have acquired the ability to recognize invariant pathogen-associated molecular patterns (PAMPs) that are characteristic of microbial organisms but which are not found in potential host plants. Such structures, also termed general elicitors of plant defense, are often indispensable for the microbial lifestyle and, upon receptor-mediated perception, inevitably betray the invader to the plant's surveillance system. Remarkable similarities have been uncovered in the molecular mode of PAMP perception in animals and plants, including the discovery of plant receptors resembling mammalian Toll-like receptors or cytoplasmic nucleotide-binding oligomerization domain leucine-rich repeat proteins. Moreover, molecular building blocks of PAMP-induced signaling cascades leading to the transcriptional activation of immune response genes are shared among the two kingdoms. In particular, nitric oxide as well as mitogen-activated protein kinase cascades have been implicated in triggering innate immune responses, part of which is the production of antimicrobial compounds. In addition to PAMP-mediated pathogen defense, disease resistance programs are often initiated upon plant-cultivar-specific recognition of microbial race-specific virulence factors, a recognition specificity that is not known from animals.