To sustain cellular homeostasis under various stress conditions, such as starvation, osmotic stress, and invading pathogens, cells need to induce a specific response. Autophagy contributes to cellular homeostasis by degrading a variety of cytoplasmic macromolecules and organelles. Autophagy is mediated by a specific double-membrane vesicle, called the autophagosome, which sequesters cytoplasmic components and delivers them to the lytic compartment. Several regulators and factors involved in autophagosome formation have been identified from yeast, mammals, and plants. Phosphatidylinositol 3-phosphate (PI3P) is one of essential phosphoinositides enriched in the multivesicular endosome and autophagosome. PI3P is generated near the site of autophagosome formation and recruits PI3P effector proteins to support autophagosome biogenesis. In Chapter 1 of this dissertation, I report the molecular function of the PI3P effector FYVE2 during autophagosome formation in Arabidopsis (Arabidopsis thaliana). FYVE2 localizes to the ER and autophagosomes, and functions downstream of ATG2-ATG18A protein complex. FYVE2 interacts with the ATG18A proteins and COPII components at the autophagosome. FYVE2 and its interaction partner SAR1B participate in a late step of autophagosome formation. To summarize, Arabidopsis FYVE2 mediates autophagosome formation by interacting with SAR1B. In Chapter 2, I propose the molecular role of Arabidopsis FYVE3 in autophagy. FYVE2 and FYVE3 have a similar domain structure but distinct subcellular distribution and protein interactions. FYVE3 localizes to the PI3P-enriched organelles, such as multivesicular endosome and autophagosome, and interacts with ATG8. fyve2 fyve3 double mutant analysis showed that fyve3 suppressed the accumulation of phagophores in fyve2 but not that of atg2. These results support the notion that FYVE3 works downstream of ATG2 but upstream of FYVE2. In total, the work described in this dissertation demonstrates the molecular functions of Arabidopsis FYVE2 and FYVE3 in autophagy.