DataSheet5_An inducible gene from glycoside hydrolase one family of Plutella xylostella decreases larval survival when feeding on host plant.PDF

Glycoside hydrolase family 1 (GH1) members exhibit a broad substrate spectrum and play important roles in insect-plant interactions, such as the defensive β-glucosidase and β-thioglucosidase (so-called myrosinase). However, knowledge about the expression profiling and function of glycoside hydrolase family 1 members in a specialist pest of crucifers Plutella xylostella is still limited. In this study, 13 putative glycoside hydrolase family 1 members of P. xylostella were identified based on the sequence characteristics, while no myrosinase activity was detectable in P. xylostella using gas chromatography-mass spectrometry (GC-MS). Expression profiling of these glycoside hydrolase family 1 members identified the midgut-specific gene Px008848 that is induced by host plant. Further experiments revealed that the in vitro expressed Px008848 protein had β-glucosidase activity and the survival rate of the larvae feeding on wounded Arabidopsis thaliana leaves declined when leaves were treated with purified Px008848 protein. When CRISPR/Cas9-based homozygous mutant larvae of Px008848 and wild-type larvae were respectively transferred onto the A. thaliana, the larval survival rate of the mutant larvae was significantly higher than that of the wild-type individuals. Our work showed that certain insect glycoside hydrolase family 1 gene may have negative effect on the development of larvae feeding on the host plant, which broadened our understandings on the evolutionary function of this gene family in the insect-plant interaction.

糖苷水解酶家族1（Glycoside hydrolase family 1, GH1）成员具有宽泛的底物谱，在昆虫-植物互作过程中发挥关键功能，例如防御性β-葡萄糖苷酶（β-glucosidase）与β-硫代葡萄糖苷酶（β-thioglucosidase，又称黑芥子酶myrosinase）。然而，当前针对十字花科专食性害虫小菜蛾（Plutella xylostella）体内糖苷水解酶家族1成员的表达谱及功能的研究认知仍较为有限。本研究基于序列特征，从小菜蛾体内鉴定得到13个推定的糖苷水解酶家族1成员；同时借助气相色谱-质谱联用（gas chromatography-mass spectrometry, GC-MS）技术检测，未在小菜蛾体内检出黑芥子酶活性。通过对上述糖苷水解酶家族1成员的表达谱分析，筛选得到一个受寄主植物诱导的中肠特异性基因Px008848。后续实验证实，体外表达的Px008848蛋白具备β-葡萄糖苷酶活性；当用纯化的Px008848蛋白处理拟南芥（Arabidopsis thaliana）损伤叶片后，取食该叶片的幼虫存活率显著下降。将基于CRISPR/Cas9构建的Px008848纯合突变体幼虫与野生型幼虫分别接种至拟南芥上饲养后，突变体幼虫的存活率显著高于野生型个体。本研究表明，部分昆虫糖苷水解酶家族1基因可能对取食寄主植物的幼虫生长发育产生负面影响，该发现拓宽了学界对该基因家族在昆虫-植物互作中进化功能的认知边界。