Yokoi Kakeru, Hayakawa Yuuki, Kato Daiki, Minakuchi Chieka, Tanaka Toshiharu, Ochiai Masanori, Kamiya Katsumi, Miura Ken
Applied Entomology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
Applied Entomology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
J Invertebr Pathol. 2015 Nov;132:190-200. doi: 10.1016/j.jip.2015.10.008. Epub 2015 Oct 28.
In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.
在本研究中，我们对赤拟谷盗的前酚氧化酶（proPO，即PPO）基因进行了表征，并研究了它们在宿主抗菌防御中的作用。与其他昆虫物种中已充分表征的PPO蛋白进行氨基酸序列比较表明，赤拟谷盗PPO基因编码功能性的酶原，关键序列基序保守。两个PPO基因PPO1和PPO2在蛹期的mRNA发育动力学彼此不同。PPO1 mRNA水平在蛹发育过程中持续下降，而PPO2的mRNA水平在蛹中期达到峰值。在用热灭活的模型微生物进行免疫挑战后，这两种mRNA也表现出不同的反应。PPO1 mRNA在挑战后6小时几乎保持不变，在24小时时略有升高。相比之下，PPO2 mRNA在挑战后3、6和24小时显著下降。无论用作诱导剂的微生物种类如何，各个PPO基因呈现的这些趋势都是一致的。最后，我们通过利用RNA干扰介导的基因沉默来研究赤拟谷盗PPO基因在宿主抗菌防御中的作用。存活试验表明，PPO基因的双重敲低伴随着血淋巴中酚氧化酶活性的减弱，显著损害了宿主对枯草芽孢杆菌的防御。相比之下，敲低对这里研究的任何赤拟谷盗抗菌肽基因的诱导没有影响，除了一个属于显示非常弱或可忽略微生物诱导的基因组。PPO敲低也适度但显著地削弱了宿主对球孢白僵菌的防御，这取决于感染方法和靶向基因的组合。我们的结果表明，PPO基因是赤拟谷盗抗菌和抗真菌宿主防御的组成部分。
