Using quantitative assays for autophagy, we analyzed 4 classes of atg mutants, discovered new atg2 phenotypes and ATG gene interactions, and proposed a model of autophagosome formation in plants. Plant and other eukaryotic cells use autophagy to target cytoplasmic constituents for degradation in the vacuole. Autophagy is regulated and executed by a conserved set of proteins called autophagy-related (ATG). In Arabidopsis, several groups of ATG proteins have been characterized using genetic approaches. However, the genetic interactions between ATG genes have not been established and the relationship between different ATG groups in plants remains unclear. Here we analyzed atg2, atg7, atg9, and atg11 mutants and their double mutants at the physiological, biochemical, and subcellular levels. Involvement of phosphatidylinositol 3-kinase (PI3K) in autophagy was also tested using wortmannin, a PI3K inhibitor. Our mutant analysis using autophagy markers showed that atg7 and atg2 phenotypes are more severe than those of atg11 and atg9. Unlike other mutants, atg2 cells accumulated several autophagic vesicles that could not be delivered to the vacuole. Analysis of atg double mutants, combined with wortmannin treatment, indicated that ATG11, PI3K, and ATG9 act upstream of ATG2. Our data support a model in which plant ATG1 and PI3K complexes play a role in the initiation of autophagy, whereas ATG2 is involved in a later step during the biogenesis of autophagic vesicles.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase SAR1, which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

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