Department of Plant Medicals, Andong National University, Andong, South Korea.
Arch Insect Biochem Physiol. 2020 Jun;104(2):e21670. doi: 10.1002/arch.21670. Epub 2020 Mar 20.
Most immune effectors are inducible to microbial pathogen infection while some are already present to act as prophylactic immunity against as yet unseen infection. This study identified secretory phospholipase A (sPLA ) as a prophylactic factor in diamondback moth, Plutella xylostella. Western blotting using a polyclonal antibody raised against other lepidopteran sPLA reacted specifically with ∼25 kDa protein, which was present at approximately 0.4 mM in the plasma of naïve larvae. Interrogation of P. xylostella transcriptomes revealed an open-reading frame for sPLA (Px-sPLA ), exhibiting high homology with other Group III sPLA s. Px-sPLA was expressed in all developmental stages. In the larval stage, bacterial challenge induced its expression in hemocytes and fat body but not in gut or epidermis. RNA interference (RNAi) suppressed Px-sPLA messenger RNA level and sPLA activity in plasma. An inhibition zone assay showed that Px-sPLA exhibited antibacterial activities against different species, because specific RNAi knockdown impaired the activity. The RNAi treatment also suppressed the cellular immune response assessed by hemocyte nodule formation and humoral immune response assessed by antimicrobial peptide gene expression. Finally, benzylideneacetone (BZA, a specific sPLA inhibitor) treatment inhibited plasma sPLA activity of naive larvae in a dose-dependent manner. An addition of BZA significantly increased the bacterial virulence of an entomopathogen, Bacillus thuringiensis. These results suggest that Px-sPLA is an immune-associated factor of P. xylostella and its relatively high level of concentration in the plasma of naive larvae strongly suggests its role as a prophylactic factor in defending against pathogens at early infection stages.
大多数免疫效应物在受到微生物病原体感染时可被诱导产生，而有些则已经存在，以作为对尚未发现的感染的预防性免疫。本研究鉴定出鳞翅目昆虫小菜蛾中的分泌型磷脂酶 A（sPLA）是一种预防性因子。使用针对其他鳞翅目 sPLA 制备的多克隆抗体进行的 Western blot 反应特异性地与约 25 kDa 的蛋白质结合，该蛋白质在未感染幼虫的血浆中约为 0.4 mM。小菜蛾转录组的分析揭示了 sPLA 的开放阅读框（Px-sPLA），它与其他第三组 sPLA 具有高度同源性。Px-sPLA 在所有发育阶段均有表达。在幼虫阶段，细菌刺激诱导其在血细胞和脂肪体中表达，但不在肠道或表皮中表达。RNA 干扰（RNAi）抑制了 Px-sPLA 信使 RNA 水平和血浆中的 sPLA 活性。抑菌圈测定表明，Px-sPLA 对不同物种具有抗菌活性，因为特异性 RNAi 敲低削弱了其活性。RNAi 处理还抑制了通过血细胞结节形成评估的细胞免疫反应和通过抗菌肽基因表达评估的体液免疫反应。最后，苯甲叉丙酮（BZA，一种特定的 sPLA 抑制剂）处理以剂量依赖的方式抑制了未感染幼虫血浆中的 sPLA 活性。BZA 的添加显著增加了昆虫病原体苏云金芽孢杆菌的细菌毒力。这些结果表明，Px-sPLA 是小菜蛾的一种免疫相关因子，其在未感染幼虫血浆中的相对高浓度强烈表明其在早期感染阶段作为防御病原体的预防性因子的作用。
