The potato cyst nematode (Globodera rostochiensis) is an obligate root pathogen of potatoes. G. rostochiensis encodes several highly expanded effector gene families including the Gr4D06 family; however, little is known about the function of this effector family. We cloned four 29D09 genes from G. rostochiensis (named Gr29D09v1/v2/v3/v4) that share high sequence similarity and are homologous to the Hg29D09 and Hg4D06 effector genes from the soybean cyst nematode. Phylogenetic analysis revealed that Gr29D09 genes belong to a subgroup of the Gr4D06 family. We showed that Gr29D09 genes are expressed exclusively within the nematode's dorsal gland cell and are dramatically upregulated in parasitic stages, indicating involvement of Gr29D09 effectors in nematode parasitism. Transgenic potato lines overexpressing Gr29D09 variants showed increased susceptibility to G. rostochiensis. Transient expression assays in Nicotiana benthamiana demonstrated that Gr29D09v3 could suppress ROS and defense gene expression induced by flg22 and cell-death mediated by immune receptors. These results suggest a critical role of Gr29D09 effectors in defense suppression. The use of affinity purification coupled with nanoliquid chromatography-tandem mass spectrometry identified potato hexokinase 1 (StHXK1) as a candidate target of Gr29D09. The Gr29D09-StHXK1 interaction was further confirmed using in planta protein-protein interaction assays. Plant HXKs have been implicated in defense regulation against pathogen infection. Interestingly, we found that StHXK1 could enhance flg22-induced ROS production, consistent with a positive role of plant HXKs in defense. Altogether, our results suggest that targeting StHXK1 by Gr29D09 effectors may impair the positive function of StHXK1 in plant immunity, thereby aiding nematode parasitism.

中文翻译：

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具有防御抑制作用的 Globodera rostochiensis Gr29D09 效应子以马铃薯己糖激酶 1 蛋白为目标。

马铃薯胞囊线虫（Globodera rostochiensis）是马铃薯的专性根病原体。G. rostochiensis 编码多个高度扩展的效应基因家族，包括 Gr4D06 家族；然而，人们对这个效应器家族的功能知之甚少。我们从 G. rostochiensis 中克隆了四个 29D09 基因（命名为 Gr29D09v1/v2/v3/v4），它们具有高度序列相似性，并且与大豆胞囊线虫的 Hg29D09 和 Hg4D06 效应基因同源。系统发育分析表明，Gr29D09 基因属于 Gr4D06 家族的一个亚组。我们发现 Gr29D09 基因仅在线虫的背腺细胞内表达，并且在寄生阶段显着上调，表明 Gr29D09 效应子参与线虫寄生。过度表达 Gr29D09 变体的转基因马铃薯品系显示出对马铃薯G. rostochiensis 的易感性增加。本塞姆氏烟草中的瞬时表达测定表明，Gr29D09v3 可以抑制 flg22 诱导的 ROS 和防御基因表达以及免疫受体介导的细胞死亡。这些结果表明 Gr29D09 效应器在防御抑制中发挥着关键作用。使用亲和纯化结合纳米液体色谱-串联质谱法将马铃薯己糖激酶 1 (StHXK1) 鉴定为 Gr29D09 的候选靶标。Gr29D09-StHXK1 相互作用通过植物蛋白质-蛋白质相互作用测定得到进一步证实。植物 HXK 与病原体感染的防御调节有关。有趣的是，我们发现 StHXK1 可以增强 flg22 诱导的 ROS 产生，这与植物 HXK 在防御中的积极作用一致。总而言之，我们的结果表明，Gr29D09 效应子靶向 StHXK1 可能会损害 StHXK1 在植物免疫中的积极功能，从而有助于线虫寄生。