ABSTRACT
In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana associated molecule with the SH3 domain of STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with endosomal complex required for transport-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit vacuolar protein sorting2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Autophagy/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Darkness , Disease Resistance/genetics , Endocytosis/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Fungi/physiology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoblotting , Microscopy, Confocal , Mutation , Plant Diseases/genetics , Plant Diseases/microbiology , Plants, Genetically Modified , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolism , Ubiquitinated Proteins/genetics , Ubiquitinated Proteins/metabolismABSTRACT
Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post-Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient-induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F-like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1-like RAB GTPase) as essential for resistance to fungal penetration into the host cell.
