Activation of microlipophagy during early infection of insect hosts by Metarhizium robertsii

The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants. Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

巨自噬/自噬机器（macroautophagic/autophagic machinery）对于丝状真菌发育与致病力的必要性已得到广泛认可，但其具体作用效应与分子机制仍未明确。昆虫病原真菌罗伯茨绿僵菌（Metarhizium robertsii）通过形成侵染结构附着胞（appressoria）穿透寄主表皮完成侵染。本研究发现，罗伯茨绿僵菌在附着胞形成过程中，自噬流（autophagic fluxes）被显著激活。
全基因组水平敲除自噬相关基因（autophagy-related, ATG）并开展昆虫生物测定后，我们从23个MrATG编码基因中筛选出10个，其缺失会导致真菌对昆虫寄主的体表侵染能力受损。除2株敲除突变体无法在昆虫体表形成附着胞外，其余致病力下降的突变体均显著受损于穿透玻璃纸膜与昆虫表皮，提示它们无法产生正常的附着胞膨压（appressorium turgor）。
我们观察到，各菌株分生孢子中的脂滴（lipid droplets, LDs）储备无显著差异，但不同突变体的自噬介导脂滴降解过程均出现受损。在通过氮饥饿诱导细胞自噬后，我们发现，在筛选出的、可能无法形成自噬体（autophagosomes）的突变体细胞中，脂滴进入液泡（vacuoles）的过程并未受到影响。上述发现揭示了该真菌中存在的微脂噬（microlipophagy）机制，且脂滴的直接吞噬过程不会受到下游脂解缺陷的抑制。
本研究首次揭示了微脂噬的激活及其在真菌侵染生物学中的作用。本研究建立的技术方法可通过检测核心自噬基因敲除突变体中液泡/溶酶体（lysosomes）对脂滴的吞噬情况，为未来在不同生物中检测微脂噬提供助力。
缩写说明：
AIM：Atg8家族相互作用基序（Atg8-family interacting motif, AIM）；
ATG：自噬相关基因（autophagy-related, ATG）；
CM：完全培养基（complete medium, CM）；
CMAC：7-氨基-4-氯甲基香豆素（7-amino-4-chloromethylcoumarin, CMAC）；
DTT：二硫苏糖醇（dithiothreitol, DTT）；
ER：内质网（endoplasmic reticulum, ER）；
GFP：绿色荧光蛋白（green fluorescent protein, GFP）；
LD：脂滴（lipid droplet, LD）；
MM：基础培养基（minimum medium, MM）；
MM-N：无氮源基础培养基（minimum medium without nitrogen source, MM-N）；
PDA：马铃薯葡萄糖琼脂（potato dextrose agar, PDA）；
PMSF：苯甲基磺酰氟（phenylmethylsulfonyl fluoride, PMSF）；
RFP：红色荧光蛋白（red fluorescent protein, RFP）；
SDB：沙氏葡萄糖肉汤（Sabouraud dextrose broth, SDB）；
SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE）；
TAG：三酰甘油（triacylglycerol, TAG）；
TEM：透射电子显微镜（transmission electron microscopy, TEM）；
WT：野生型（wild type, WT）。