Functional identification of C-type lectin in the diamondback moth, Plutella xylostella (L.) innate immunity

doi:10.1016/S2095-3119(21)63650-X

摘要/Abstract

摘要：

C-型凝集素(C-type lectins，CTLs)是一种依赖Ca²⁺的碳水化合物识别蛋白超家族，也是昆虫天然免疫中一类重要的模式识别受体(pattern recognition receptor，PRR)，可介导昆虫的体液免疫和细胞免疫。我们从小菜蛾基因组中克隆到了一个新的含有双碳水化合物识别结构域(carbohydrate-recognition domain，CRD)的C-型凝集素，命名为PxIML。PxIML的开放阅读框长969 bp，编码322个氨基酸残基。该蛋白序列含有一个信号肽和一个带有EPN(Glu₁₂₄-Pro₁₂₅-Asn₁₂₆)和QPD(Gln₂₇₄-Pro₂₇₅-Asp₂₇₆)基序的双碳水化合物识别结构域。表达谱分析发现，PxIML在脂肪体中的表达显著高于血淋巴和中肠。苏云金芽胞杆菌(Bacillus thuringiensis-8010，Bt8010)感染小菜蛾18 h后，PxIML在全虫和脂肪体中的表达受到抑制，而被粘质沙雷氏菌IAE6(Serratia marcescens-IAE6)和毕赤酵母(Pichia pastoris)感染后，PxIML在全虫和脂肪体中的表达显著上升。重组PxIML(rPxIML)能与所测试的病原体相关分子模式(pathogen-associated molecular patterns，PAMPs)，以及肠杆菌IAE5(Enterobacter sp. IAE5)、粘质沙雷氏菌IAE6、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌BL21(Escherichia coli BL21)和Bt8010以依赖Ca²⁺的方式结合，但与毕赤酵母不结合。在Ca²⁺存在的条件下，rPxIML对Bt8010、肠杆菌IAE5和金黄色葡萄球具有较强的凝集活性，但对大肠杆菌BL21的凝集活性弱，对粘质沙雷氏菌IAE6和毕赤酵母不凝集。此外，rPxIML还能与血细胞结合，促进血细胞对包被rPxIML凝胶珠的吸附，增强酚氧化酶活性，促进黑化反应。我们的研究结果表明，PxIML在病原体识别和介导小菜蛾随后的体液免疫和细胞免疫中起着重要作用。

Abstract:

C-type lectins (CTLs) are a superfamily of Ca²⁺-dependent carbohydrate-recognition proteins, and an important pattern recognition receptor (PRR) in insect innate immunity which can mediate humoral and cellular immunity in insects. In this study, we report a novel dual carbohydrate-recognition domain (CRD) CTL from Plutella xylostella which we designate PxIML. PxIML is a protein with a 969 bp open reading frame (ORF) encoding 322 amino acids, containing a signal peptide and a dual-CRD with EPN (Glu₁₂₄-Pro₁₂₅-Asn₁₂₆) and QPD (Gln₂₇₄-Pro₂₇₅-Asp₂₇₆) motifs. The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut. The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010 (Bt8010) at 18 h, while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris. The recombinant PxIML (rPxIML) protein could bind to the tested pathogen-associated molecular patterns (PAMPs), and the bacteria of Enterobacter sp. IAE5, S. marcescens IAE6, Staphylococcus aureus, Escherichia coli BL21, and Bt8010 in a Ca²⁺-dependent manner, however, it showed limited binding to the fungus, P. pastoris. The rPxIML exhibited strong activity in the presence of Ca²⁺ to agglutinate Bt8010, Enterobacter sp. IAE5 and S. aureus, but it only weakly agglutinated with E. coli BL21, and could not agglutinate with S. marcescens IAE6 or P. pastoris. Furthermore, the rPxIML could bind to hemocytes, promote the adsorption of hemocytes to beads, and enhance the phenoloxidase (PO) activity and melanization of P. xylostella. Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P. xylostella.

Key words:

diamondback moth , humoral and cellular immunity , C-type lectin , pattern recognition receptors , carbohydrate-recognition domain

. [J]. Journal of Integrative Agriculture, 2021, 20(12): 3240-3255.

LI Jin-yang, LIN Jun-han, G. Mandela FERNáNDEZ-GRANDON, ZHANG Jia-yu, YOU Min-sheng, XIA Xiao-feng. Functional identification of C-type lectin in the diamondback moth, Plutella xylostella (L.) innate immunity[J]. Journal of Integrative Agriculture, 2021, 20(12): 3240-3255.

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