Clathrin light chains negatively regulate plant immunity by hijacking the autophagy pathway

Area of Science:

• Plant cell biology
• Molecular plant science
• Endocytosis and autophagy research

Background:

• Crosstalk between clathrin-mediated endocytosis (CME) and autophagy is known in mammals but unestablished in plants.
• Autophagy-related genes (ATGs) are crucial for autophagy.
• Clathrin light chains (CLCs) are components of the CME machinery.

Purpose of the Study:

• To investigate the interconnection between CME and autophagy in plants.
• To determine if CLCs play a role in the autophagy pathway in Arabidopsis.
• To elucidate the molecular mechanisms linking CLCs and autophagy.

Main Methods:

• Generation and analysis of Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutants (clc2-1 clc3-1).
• Phenotypic analysis including autoimmunity and nutrient sensitivity assays.
• Biochemical assays to investigate protein-protein interactions (e.g., CLC2 with ATG8h/ATG8i).
• Autophagic degradation assays using fluorescently tagged proteins (GFP-ATG8h, GFP-ATG8i, CLC2-GFP).
• CRISPR-Cas9 gene editing to generate ATG8h and ATG8i knockout mutants.

Main Results:

• The clc2-1 clc3-1 mutant exhibits phenotypes similar to ATG mutants, indicating compromised autophagy.
• CLC2 directly interacts with ATG8h and ATG8i in a domain-specific manner.
• Autophagic degradation of GFP-ATG8h/GFP-ATG8i and CLC2-GFP is observed, with reduced GFP-ATG8h degradation in the clc2-1 clc3-1 mutant.
• Simultaneous knockout of ATG8h and ATG8i confers enhanced resistance to Golovinomyces cichoracearum.

Conclusions:

• This study reveals a functional link between CLCs and the autophagy pathway in Arabidopsis.
• CLC2 is a novel component involved in regulating autophagy.
• The interaction between CLC2 and ATG8 proteins is functionally relevant for plant immunity.