A facile forward-genetic screen for <i>Arabidopsis</i> autophagy mutants reveals twenty-one loss-of-function mutations disrupting six <i>ATG</i> genes

Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (<i>ATG</i>) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved <i>ATG</i> genes in plants, only a few <i>atg</i> mutants have emerged from forward-genetic screens in <i>Arabidopsis thaliana</i>. We developed a screen that consistently recovers <i>Arabidopsis atg</i> mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. <i>Arabidopsis lon2</i> mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a <i>lon2</i> suppressor screen to expedite recovery of additional <i>atg</i> mutants. After screening mutagenized <i>lon2-2</i> seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 <i>ATG</i> genes: <i>ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230</i>, and <i>ATG18a/At3g62770</i>. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional <i>atg11</i>/<i>At4g30790</i> allele incompletely suppresses <i>lon2</i> defects. Finally, we show that NBR1 is not necessary for autophagy of <i>lon2</i> peroxisomes and that <i>NBR1</i> overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that <i>Arabidopsis</i> can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. <b>Abbreviations:</b> ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type