Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidopsis thaliana) NBR1 (next to BRCA1 gene 1 protein; AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a ubiquitin-associated (UBA)-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

选择性自噬是一种亚细胞过程，细胞质物质被选择性地隔离到自噬体中，随后输送到液泡进行降解和回收。拟南芥 ( Arabidopsis thaliana ) NBR1（紧邻 BRCA1 基因 1 蛋白；AtNBR1）已被认为在植物中充当选择性自噬受体，AtNBR1 将泛素化靶标锚定到自噬体上进行降解。然而，AtNBR1 的具体货物仍然难以捉摸。我们之前表明，拟南芥外囊亚基 EXO70 家族蛋白 E2 (AtExo70E2) 是外囊阳性细胞器 (EXPO) 的标记，与自噬体标记拟南芥自噬相关蛋白 8 (AtATG8) 共定位，并在自噬诱导后被递送至液泡进行降解。在这里，通过多重分析，我们证明 AtNBR1 是拟南芥自噬过程中 AtExo70E2 的选择性受体。首先，两种新型的功能缺失型nbr1 CRISPR 突变体（ nbr1 - c1和nbr1 - c2 ）在短日照生长条件下表现出早衰老表型。其次，在自噬​​诱导过程中，与野生型植物相比， nbr1突变体中 AtExo70E2 或 EXPO 的液泡递送显着减少。第三，生化和招募测定表明，AtNBR1 在自噬过程中以不依赖于泛素相关 (UBA) 的方式与 AtATG8 阳性自噬体特异性相互作用并将 AtExo70E2 或其 EXPO 招募到 AtATG8 阳性自噬体中。综上所述，我们的数据表明，AtNBR1 作为选择性受体，在植物自噬中以不依赖于 UBA 的方式介导 AtExo70E2 或 EXPO 的液泡传递。