沉默囊泡分选蛋白(VPS)对辣椒疫霉效应因子Rx LR504功能的影响

Effect of Transient Silencing of Vacuolar Sorting Protein on the Function of Phytophthora capsici RxLR504

辣椒疫霉(Phytophthora capsici)是一种重要的病原卵菌,能产生大量的RxLR效应分子帮助病原菌定殖。前期实验证明RxLR504效应分子能够抑制INF1引发的细胞死亡,并证实囊泡分选蛋白(VPS)与其相互作用。本文借助VIGS tool在线工具设计靶标基因沉默片段;利用烟草脆裂病毒(TRV)介导的基因沉默技术(VIGS)沉默本氏烟靶标基因;提取沉默样品叶片组织RNA,构建c DNA文库;利用qRT-PCR检测靶标基因表达水平;在沉默植株叶片上表达RxLR504,24 h后接种INF1,观察病斑症状。结果表明:本氏烟中设计沉默片段长度为300 bp,构建c DNA文库质量较高;荧光定量PCR结果显示,沉默植株中靶标基因表达水平下降86.2%,与对照相比差异性显著;接种实验证明RxLR504在沉默植株与对照植株上均能抑制INF1引发的细胞死亡。RxLR504与VPS互作不影响其抑制INF1引起的细胞坏死,为进一步揭示病原物利用植物靶标基因抑制植物免疫从而促进自身寄生的机制打下基础。

Phytophthora capsici is an important pathogenic oomycetes, which produces a large number of RxLR effectors to help the pathogen to colonize. However, the mechanism of RxLR effector in Phytophthora capsici is relatively less studied. It was previously demonstrated that RxLR504 effector could inhibit INF1-induced cell death, and interacts with vesicle sorting proteins(VPS). Online VIGS tool was used to design target fragments. Gene silencing mediated by tobacco fragile cracking virus(TRV) was used to silence target gene in N.benthamiana. RNA was extracted from leaf tissue of silenced samples to construct c DNA library. The expression level of target gene was detected by qRT-PCR. INF1 was inoculated 24 hours after inoculation with effector RxLR504 to observe the symptoms of pots. The results show that the length of the targeted silence segment is 300 bp and the quality of the constructed c DNA library is pure. Fluorescence quantitative results showed that the expression level of target genes in the silenced plants decreased by 86.2%, which was significantly different from the control.Inhibition experiments demonstrated that RxLR504 could inhibit INF1-induced cell death in both silencing and control plants.The experiments proved that the interaction of RxLR504 and VPS did not affect the inhibition of cell necrosis caused by INF1, which laid a foundation for further revealing the mechanism of pathogen inhibiting plant immunity by regulating plant target genes to promote its own parasitism.