A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.

中文翻译：

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M35家族的金属蛋白酶是通过灭活宿主酚氧化酶及其他物质来抵抗昆虫的真菌毒力所必需的。

真核生物中分布着多种金属蛋白酶（MPs）。但是，国会议员的职能仍在研究中。我们报告说，属于M35家族的7个MP在昆虫致病性真菌罗氏沼虾的基因组中编码。通过基因删除和昆虫生物测定，我们发现其中一个M35家族的MP，即MrM35-4，对于抵抗昆虫宿主的真菌毒力是必需的。MrM35-4是一种可分泌的酶，其蛋白水解活性与促进昆虫表皮的真菌渗透有关。基因拯救和过表达后，昆虫生物测定表明，MrM35-4有助于抑制昆虫的表皮和血细胞黑色素化活性。酶促裂解分析显示，MrM35-4可以将黑腹果蝇的重组前酚氧化酶PPO1和PPO2以不同于可以激活PPO活性的丝氨酸蛋白酶的方式夹住。另外，发现MrM35-4与抑制昆虫中的抗真菌基因表达有关。与MrM35-4对宿主细胞的明显凋亡作用相一致，我们发现PPO突变体以不同的方式死于罗伯茨曼氏菌的野生型和突变株的感染。因此，MrM35-4在真菌-昆虫相互作用过程中除了靶向PPO以外，还发挥着多方面的作用，这代表了原发菌所采用的先前未曾预料到的策略，可以胜过昆虫的免疫防御。发现MrM35-4与抑制昆虫中的抗真菌基因表达有关。与MrM35-4对宿主细胞的明显的凋亡作用相一致，我们发现PPO突变体以不同的方式死于罗伯茨氏菌的野生型和突变株的感染。因此，MrM35-4在真菌-昆虫相互作用过程中除了靶向PPO以外，还发挥着多方面的作用，这代表了原发菌所采用的先前未曾预料到的策略，可以胜过昆虫的免疫防御。发现MrM35-4与抑制昆虫中的抗真菌基因表达有关。与MrM35-4对宿主细胞的明显凋亡作用相一致，我们发现PPO突变体以不同的方式死于罗伯茨曼氏菌的野生型和突变株的感染。因此，MrM35-4在真菌-昆虫相互作用过程中除了靶向PPO以外，还发挥着多方面的作用，这代表了原发菌所采用的先前未曾预料到的策略，可以胜过昆虫的免疫防御。