The role of selective autophagy in growth and development of filamentous fungi

在丝状子囊菌中，自噬参与了多种发育过程。有两种类型的自噬被描述：非选择性和选择性自噬。非选择性自噬是细胞质和细胞器随机吞噬到双膜囊泡中，自噬体将货物运送到液泡中进行降解。在选择性自噬中，特定的货物如细胞器、蛋白质聚集体或酶被货物受体识别并包裹在自噬体中。对于有性繁殖，丝状真菌产生三维子实体，在那里产生性子囊孢子。同源子囊菌大孢子Sordaria macrospora是研究多细胞子实体发育的优良真菌模式生物。近年来，已经建立了分子遗传学方法来分离和表征大孢子葡萄球菌的发育基因；然而，自噬在多细胞子实体发育中的确切作用在很大程度上是未知的。利用反向遗传学方法，我们之前已经表明，在大孢子葡萄子实体发育中，选择性和非选择性自噬机制核心功能所需的保守基因是必不可少的。我们使用GFP-Trap分析与泛素样膜相关的egfp标记的SmATG8来鉴定相互作用的蛋白。我们的研究确定了17个蛋白作为假定的SmATG8相互作用伙伴，其中一个是假定的哺乳动物BRCA1的选择性货物受体邻居（NBR1）的同源蛋白。在哺乳动物和植物中，NBR1被证明是泛素化底物自噬降解和过氧化物酶体降解的货物受体或适配器。酿酒酵母中没有编码NBR1的同源物；因此，大孢子葡萄球菌是研究这种自噬受体功能的一个有用模型。双杂交实验、共免疫沉淀和体内共定位表明，SmNBR1和SmATG8可以直接相互作用。此外，我们可以证明SmNBR1以依赖于smatg8的方式转运到液泡。DeltaSmnbr1突变体在饥饿条件下生长速度下降，性发育延迟，以及子囊孢子形成和食腐功能受损。通过研究这两种蛋白的相互作用，并确定SmATG8和SmNBR1的新相互作用伙伴，我们旨在了解选择性自噬如何促进真菌的营养生长和发育。

In filamentous ascomycetes, autophagy is involved in various developmental processes. Two types of autophagy have been described: non-selective and selective autophagy. Non-selective autophagy is the random engulfment of cytoplasm and organelles into double-membrane vesicles, the autophagosomes, which deliver the cargo to the vacuole for degradation. In selective autophagy, specific cargos such as organelles, protein aggregates or enzymes are recognized by cargo receptors and enwrapped into autophagosomes. For sexual propagation, filamentous fungi produce three-dimensional fruiting bodies, where the sexual ascospores are generated. The homothallic (self-fertile) ascomycete Sordaria macrospora is an excellent fungal model organism to study multicellular fruiting-body development. In recent years, molecular genetics procedures have been established to isolate and characterize developmental genes in S. macrospora; however, the exact role of autophagy in multicellular fruiting-body development is largely unknown.Using a reverse genetics approach, we have previously shown that conserved genes required for core functions of the selective and non-selective autophagic machinery are essential for fruiting-body development in S. macrospora. We used GFP-Trap analysis with the ubiquitin-like membrane associated EGFP-tagged SmATG8 to identify interacting proteins. Our study identified 17 proteins as putative SmATG8 interaction partners, among these a putative homolog of the mammalian selective cargo receptor Neighbor of BRCA1 (NBR1). In mammals and plants, NBR1 was shown to act as cargo receptor or adaptor for the autophagic degradation of ubiquitinated substrates and the degradation of peroxisomes. A homolog of NBR1 is not encoded in Saccharomyces cerevisiae; therefore S. macrospora is a useful model to study the functions of this autophagy receptor. Two-hybrid experiments, co-immunoprecipitation and in vivo co-localization revealed that SmNBR1 and SmATG8 can directly interact with each other. In addition, we could show that SmNBR1 is transported to the vacuole in a SmATG8-dependent manner. A DeltaSmnbr1 mutant revealed a decreased growth rate under starvation conditions and a delayed sexual development as well as impairment in ascospore formation and pexophagy. By studying the interaction of both proteins and identifying new interaction partners of SmATG8 and SmNBR1, we aim to understand how selective autophagy contributes to vegetative growth and development in fungi.