Journal of Advanced Research ( IF 11.4 ) Pub Date : 2022-07-22 , DOI: 10.1016/j.jare.2022.07.004 Jie Zhao 1 , Shiming Liu 1
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Introduction
Plant parasitic cyst nematodes secrete a number of effectors into hosts to initiate formation of syncytia and infection causing huge yield losses.
Objectives
The identified cyst nematode effectors are still limited, and the cyst nematode effectors-involved interaction mechanisms between cyst nematodes and plants remain largely unknown.
Methods
The t-SNARE domain-containing effector in beet cyst nematode (BCN) was identified by In situ hybridization and immunohistochemistry analyses. The mutant of effector gene was designed by protein structure modeling analysis. The functions of effector gene and its mutant were analyzed by genetic transformation in Arabidopsis and infection by BCN. The protein–protein interaction was analyzed by yeast two hybrid, BiFC and pulldown assays. Gene expression was assayed by quantitative real-time PCR.
Results
A t-SNARE domain-containing BCN HsSNARE1 was identified as an effector, and its mutant HsSNARE1-M1 carrying three mutations (E141D, A143T and −148S) that altered regional structure from random coils to α-helixes was designed and constructed. Transgenic analyses indicated that expression of HsSNARE1 significantly enhanced while expression of HsSNARE1-M1 and highly homologous HgSNARE1 remarkably suppressed BCN susceptibility of Arabidopsis. HsSNARE1 interacted with AtSNAP2 and AtPR1 via its t-SNARE domain and N-terminal, respectively, while HsSNARE1-M1/HgSNARE1 could not interact with AtPR1 but bound AtSNAP2. AtSNAP2, AtSHMT4 and AtPR1 interacted pairwise, but neither HsSNARE1 nor HsSNARE1-M1/HgSNARE1 could interact with AtSHMT4. Expression of HsSNARE1 significantly suppressed while expression of HsSNARE1-M1/HgSNARE1 considerably induced both AtSHMT4 and AtPR1 in transgenic Arabidopsis infected with BCN. Overexpression of AtPR1 significantly suppressed BCN susceptibility of Arabidopsis.
Conclusions
This work identified a t-SNARE-domain containing cyst nematode effector HsSNARE1 and deciphered a molecular mode of action of the t-SNARE-domain containing cyst nematode effectors that HsSNARE1 promotes cyst nematode disease by interaction with both AtSNAP2 and AtPR1 and significant suppression of both AtSHMT4 and AtPR1, which is mediated by three structure change-causing amino acid residues.
中文翻译：
甜菜囊肿线虫 HsSNARE1 与 AtSNAP2 和 AtPR1 相互作用并促进拟南芥疾病
介绍
植物寄生胞囊线虫将许多效应物分泌到宿主中以启动合胞体的形成和感染，从而导致巨大的产量损失。
目标
已鉴定的胞囊线虫效应物仍然有限，并且胞囊线虫效应物与胞囊线虫和植物之间的相互作用机制在很大程度上仍然未知。
方法
甜菜胞囊线虫 (BCN) 中包含t -SNARE结构域的效应子通过原位杂交和免疫组织化学分析鉴定。通过蛋白质结构建模分析设计效应基因的突变体。通过对拟南芥的遗传转化和BCN感染，分析了效应基因及其突变体的功能。通过酵母二杂交、BiFC 和 pulldown 测定分析蛋白质-蛋白质相互作用。通过定量实时PCR测定基因表达。
结果
含有t - SNARE 结构域的 BCN HsSNARE1 被确定为效应子，其突变体 HsSNARE1-M1 携带三个突变（E141D、A143T 和 −148S），将区域结构从随机卷曲改变为 α-螺旋。转基因分析表明，HsSNARE1的表达显着增强，而HsSNARE1-M1和高度同源的HgSNARE1 的表达显着抑制拟南芥的 BCN 易感性。HsSNARE1 通过其t -SNARE 域和N与 AtSNAP2 和 AtPR1 相互作用-终端，而 HsSNARE1-M1/HgSNARE1 不能与 AtPR1 交互但绑定 AtSNAP2。AtSNAP2、AtSHMT4 和 AtPR1 成对相互作用，但 HsSNARE1 和 HsSNARE1-M1/HgSNARE1 均不能与 AtSHMT4 相互作用。在感染了 BCN 的转基因拟南芥中，HsSNARE1的表达显着受到抑制，而HsSNARE1-M1/HgSNARE1的表达显着诱导了AtSHMT4和AtPR1 。AtPR1的过表达显着抑制了拟南芥的 BCN 敏感性。
结论
这项工作确定了一个包含胞囊线虫效应子 HsSNARE1 的t -SNARE 结构域，并破译了包含胞囊线虫效应子的t -SNARE 结构域的分子作用模式，即 HsSNARE1 通过与 AtSNAP2 和 AtPR1 相互作用并显着抑制两者促进胞囊线虫病AtSHMT4和AtPR1，由三个引起结构变化的氨基酸残基介导。
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