The brown planthopper (BPH), Nilaparvata lugens (Stål), is a phloem sap-feeding insect. During feeding on rice plants, BPH secretes salivary proteins with potential effector functions, which may play a critical role in the plant-insect interactions. However, a limited number of BPH effector proteins have been identified to date. Here, we sequenced the salivary gland transcriptomes of five BPH populations and subsequently established a N. lugens secretome consisting of 1,140 protein-encoding genes. Secretome analysis revealed the presence of both conserved and rapidly evolving salivary proteins. A screen for potential effectors that elicit responses in the plant was performed via the transient expression analysis of 64 BPH salivary proteins in Nicotiana benthamiana leaves and rice protoplasts. The salivary proteins Nl12, Nl16, Nl28, and Nl43 induced cell death, whereas Nl40 induced chlorosis and Nl32 induced a dwarf phenotype in N. benthamiana, indicating effector properties of these proteins. Ectopic expression of the six salivary proteins in N. benthamiana upregulated expression of defense-related genes and callose deposition. Tissue expression analysis showed a higher expression level of the six candidate effectors in salivary glands than in other tissues. Subcellular localization and analysis of the domain required for cell death showed a diverse structure of the six effectors. Nl28, Nl40, and Nl43 are N. lugens specific; in contrast, Nl12, Nl16, and Nl32 are conserved among insects. The Nl40 family has numerous isoforms produced by alternative splicing, exemplifying rapid evolution and expansion of effector proteins in the BPH. Our results suggest a potential large effector repertoire in BPH and a higher level of effector conservation exist in BPH compared with that in plant pathogens.

中文翻译：

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唾液蛋白的分泌组分析和植物内表达鉴定了褐飞虱 Nilaparvata lugens 的候选效应子。


褐飞虱 (BPH) Nilaparvata lugens (Stål) 是一种以韧皮部汁液为食的昆虫。在以水稻为食期间，褐飞虱会分泌具有潜在效应功能的唾液蛋白，这可能在植物与昆虫的相互作用中发挥关键作用。然而，迄今为止，已鉴定出数量有限的 BPH 效应蛋白。在这里，我们对五个 BPH 群体的唾液腺转录组进行了测序，随后建立了由 1,140 个蛋白质编码基因组成的褐飞虱分泌组。分泌组分析揭示了保守且快速进化的唾液蛋白的存在。通过对本塞姆氏烟草叶子和水稻原生质体中 64 种 BPH 唾液蛋白的瞬时表达分析，对在植物中引起反应的潜在效应子进行了筛选。唾液蛋白 Nl12、Nl16、Nl28 和 Nl43 诱导细胞死亡，而 Nl40 诱导失绿，Nl32 诱导本塞姆氏烟草矮化表型，表明这些蛋白的效应子特性。本塞姆氏烟草中六种唾液蛋白的异位表达上调了防御相关基因和胼胝质沉积的表达。组织表达分析显示，唾液腺中六种候选效应器的表达水平高于其他组织。细胞死亡所需结构域的亚细胞定位和分析显示了六种效应子的不同结构。 Nl28、Nl40 和 Nl43 是褐飞虱特有的；相反，Nl12、Nl16 和 Nl32 在昆虫中是保守的。 Nl40家族具有通过选择性剪接产生的许多同工型，例证了BPH中效应蛋白的快速进化和扩展。 我们的结果表明，与植物病原体相比，BPH 中存在潜在的大量效应子库，并且 BPH 中存在更高水平的效应子保守性。