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, 15 (6), 941-959

A Facile Forward-Genetic Screen for Arabidopsis Autophagy Mutants Reveals Twenty-One Loss-Of-Function Mutations Disrupting Six ATG Genes

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A Facile Forward-Genetic Screen for Arabidopsis Autophagy Mutants Reveals Twenty-One Loss-Of-Function Mutations Disrupting Six ATG Genes

Pierce G Young et al. Autophagy.

Abstract

Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 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 lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 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 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. 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 atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. Abbreviations: 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.

Keywords: LON2 protease; organelle quality control; peroxisome turnover; pexophagy; suppressor genetics.

Figures

Figure 1.
Figure 1.
Four-step strategy for isolating Arabidopsis autophagy-defective mutants. (a) Initial screening for lon2 suppressors. lon2 forms few lateral roots in response to IBA; putative suppressors that formed lateral roots in the presence of IBA similar to wild-type and lon2-2 atg7-4 were moved to soil for propagation. Top panel: wild-type (WT), lon2-2, and lon2-2 atg7-4 seedlings were grown on media containing 8 µM IBA and imaged at 8 days; scale bar: 5 mm. Bottom row: magnified images of roots outlined in the top panel showing lateral roots, which are absent in lon2-2; scale bar: 1 mm. (b) Secondary screening of putative suppressors. Leaf extracts from approximately 30-day-old adult controls (left of dashed line) or M2 putative suppressor plants (right of dashed line) were processed for immunoblotting with antibodies to the indicated proteins. NBR1 is a selective autophagy receptor that accumulates in atg mutants [14]. PMDH is synthesized as a precursor (p) that is processed to a mature form (m) in the peroxisome. HSC70 is a loading control. The asterisk indicates a protein cross-reacting with the ATG7 antibody. (c) Retesting progeny of putative suppressors that displayed restored PTS2 processing in the M2 generation. Top: lateral root density of 8-day-old controls (left of dashed line) or M3 or M4 suppressor seedlings (right of dashed line) grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. Bottom: extracts from 6-day-old controls (left of dashed line) or M3 or M4 suppressor seedlings (right of dashed line) were processed for immunoblotting. Membranes from duplicate gels were serially probed with antibodies to the indicated proteins to obtain the top 3 and bottom 4 panels. MLS, thiolase, and ICL are peroxisomal proteins that are stabilized when both LON2 and autophagy are defective [48]. Asterisks indicate proteins cross-reacting with the ATG7 or ICL antibodies. (d) Identifying mutations via whole-genome sequencing. The L40 suppressor was backcrossed to the original lon2-2 line, IBA-sensitive F2 seedlings were selected, and genomic DNA from pooled F3 seedlings was sequenced. Homozygous single-nucleotide polymorphisms consistent with EMS mutagenesis (G/C to A/T transitions) and causing nonsynonymous mutations in coding regions, altering splice sites, or occurring in introns or untranslated regions are indicated by locus identifiers and displayed to the right of the 5 Arabidopsis chromosomes using The Arabidopsis Information Resource Chromosome Map Tool.
Figure 2.
Figure 2.
Numerous novel atg7 alleles recovered as lon2 suppressors. (a) Diagram of the ATG7 gene. Boxes and lines represent protein-coding regions and introns, respectively. The ThiF-like adenylation domain and active-site Cys residue are indicated. The positions of new atg7 mutations identified as lon2 suppressors are in blue; previously described EMS-derived lon2 suppressors [48] are in gray, and T-DNA insertion alleles [8,9,61] are indicated by triangles. atg7-9 is an unpublished allele (alias 8–30) from the pilot lon2-2 suppressor screen [48] that carries a g2959a mutation in the intron 10 splice acceptor site. The sequence of the atg7-13 nonsense allele compared to atg7-5 and wild-type ATG7 is shown below the gene diagram. aa, amino acids. (b) Lateral root density of 8-day-old wild type (WT), lon2-2, atg7-4, and lon2-2 atg7 seedlings grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. (c) Extracts from 6-day-old seedlings were processed for immunoblotting with antibodies to the indicated proteins. The asterisk indicates a protein cross-reacting with the ATG7 antibody.
Figure 3.
Figure 3.
Novel atg3, atg5, and atg16 alleles recovered as lon2 suppressors. (a) Diagrams of the ATG3, ATG5, and ATG16 genes. Boxes and lines represent protein-coding regions and introns, respectively. The positions of new atg3, atg5, and atg16 mutations identified as lon2 suppressors are shown in pink, red, and orange, respectively; the previously described atg3-1 EMS-derived lon2 suppressor [48] is in gray, and T-DNA insertion alleles [29,72,73] are indicated by triangles. aa, amino acids. (b) Lateral root density of 8-day-old wild type (WT), lon2-2, atg3-1, lon2-2 atg3-2, lon2-2 atg5, and lon2-2 atg16 seedlings grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. (c) Extracts from 6-day-old seedlings were processed for immunoblotting. Membranes from duplicate gels were serially probed with antibodies to the indicated proteins to obtain the top 5 and bottom 3 panels. The asterisk indicates a protein cross-reacting with the ATG5 antibody. (d) Extracts from 6-day-old seedlings were processed for immunoblotting and serially probed with antibodies to NBR1, ATG8A, and HSC70. ATG8 is lipidated and encoded by multiple genes in Arabidopsis.
Figure 4.
Figure 4.
Novel atg2 and atg18a alleles recovered as lon2 suppressors. (a) The L60 suppressor was backcrossed to the original lon2-2 line, and genomic DNA from pooled IBA-sensitive F2 seedlings was sequenced and analyzed as in the legend to Figure 1(d). (b) ATG2 gene diagram with boxes and lines representing protein-coding regions and introns, respectively. The positions of new atg2 mutations identified as lon2 suppressors are shown in green, previously described EMS-derived lon2 suppressors [48] are in gray, and a T-DNA insertion allele [64] is indicated by a triangle. The partial alignment shows predicted Brassicaceae ATG2 proteins, including 2 A. thaliana ATG2 splice variants predicted by the latest genome annotation (Araport 11); the alternative ATG2 splice variant is interrupted by the atg2-6 mutation. aa, amino acids. (c) ATG18a gene diagram showing atg18a mutations identified as lon2 suppressors (teal) and a previously described T-DNA insertion allele [65] (triangle). (d) Lateral root density of 8-day-old wild type (WT), lon2-2, atg2-4, lon2-2 atg2, and lon2-2 atg18a seedlings grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. (e) Extracts from 6-day-old seedlings were processed for immunoblotting with antibodies to the indicated proteins.
Figure 5.
Figure 5.
Increased peroxisome abundance in atg18a-3 seedlings. (a) Cotyledon epidermal cells, (b) cotyledon mesophyll cells, and (c) hypocotyl cells in light-grown 4-, 5-, 6-, and 7-day-old wild-type and twice backcrossed atg18a-3 seedlings expressing the peroxisomal matrix marker GFP-PTS1 were imaged for GFP fluorescence (green) and chlorophyll autofluorescence (magenta) using confocal microscopy. Scale bars: 20 µm.
Figure 6.
Figure 6.
Loss of ATG11 partially suppresses lon2 defects. (a) Diagram of the ATG11 gene. Boxes and lines represent protein-coding regions and introns, respectively. A triangle marks the location of the atg11-1 T-DNA insertion allele [17]. aa, amino acids. (b) Lateral root density of 8-day-old wild type (WT), lon2-2, atg7-4, atg11-1, lon2-2 atg7-4, and lon2-2 atg11-1 seedlings grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. (c) Extracts from 4-, 6-, and 8-day-old seedlings were processed for immunoblotting with antibodies to the indicated proteins. An asterisk indicates a protein cross-reacting with the NBR1 antibody.
Figure 7.
Figure 7.
NBR1 is not necessary for pexophagy of lon2 peroxisomes, and excess NBR1 is not sufficient to induce pexophagy. (a) Diagram of the NBR1 gene. Boxes and lines represent protein-coding regions and introns, respectively. The PB1 domain, zinc finger, NBR1 domain, ATG8 binding site, and ubiquitin-associated (UBA) domains are indicated. Triangles mark the locations of T-DNA insertion alleles. aa, amino acids. (b) Lateral root density of 8-day-old wild type (WT), lon2-2, nbr1, atg7-3, lon2-2 nbr1, lon2-2 atg7-4, 35S:NBR1, 35S:HA-NBR1, and 35S:YFP-NBR1 seedlings grown without or with IBA. Error bars show standard deviations (n = 8). Statistically significant (P < 0.0001) differences determined by one-way ANOVA are depicted by different letters above the bars. (c) Extracts prepared from 4- and 6-day-old seedlings were processed for immunoblotting with antibodies to the indicated proteins. (d) Hypocotyl cells in 6-day-old wild-type, atg18a-3, and nbr1-4 light-grown seedlings expressing the peroxisomal matrix marker GFP-PTS1 were imaged for GFP fluorescence using confocal microscopy. Scale bar: 20 µm. (e) Extracts prepared from 4- and 6-day-old seedlings were processed for immunoblotting with antibodies to the indicated proteins.

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