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

在丝状子囊菌中，自噬参与各种发育过程。自噬有两种类型：非选择性自噬和选择性自噬。非选择性自噬是细胞质和细胞器被随机吞噬到双膜囊泡（自噬体）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进入自噬体。对于有性繁殖，丝状真菌产生三维子实体，其中产生有性子囊孢子。同宗配合（自花受精）子囊菌大孢球孢菌（Sordaria macrospora）是研究多细胞子实体发育的理想模式真菌。近年来，分子遗传学方法已经建立，分离和表征的发育基因在S。大孢子虫;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。使用反向遗传学方法，我们以前已经表明，选择性和非选择性自噬机制的核心功能所需的保守基因是必不可少的子实体发育在S。大孢子虫。我们使用GFP-Trap分析与泛素样膜相关的EGFP标记的SmATG 8，以确定相互作用的蛋白质。我们的研究确定了17种蛋白质作为假定的SmATG 8相互作用伙伴，其中一个假定的同源物的哺乳动物选择性货物受体邻居BRCA 1（NBR 1）。在哺乳动物和植物中，NBR 1被证明作为货物受体或适配器，用于自噬降解泛素化底物和降解过氧化物酶体。NBR 1的同源物在酿酒酵母中不编码，因此S.大孢子虫是研究这种自噬受体功能的有用模型。双杂交、免疫共沉淀和体内共定位实验表明SmNBR 1和SmATG 8可以直接相互作用。此外，我们可以表明，SmNBR 1运输到液泡中的SmATG 8依赖的方式。一个DeltaSmnbr 1突变体显示了饥饿条件下的生长速度下降，性发育延迟，以及在子囊孢子形成和pexophagy的损害。通过研究这两种蛋白质的相互作用，并确定SmATG 8和SmNBR 1的新的相互作用伙伴，我们的目标是了解选择性自噬如何有助于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.