Chen I-Hsuan, Huang Ying-Ping, Tseng Ching-Han, Ni Jian-Tang, Tsai Chung-Han, Hsu Yau-Heiu, Tsai Ching-Hsiu
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
Front Plant Sci. 2017 Oct 6;8:1736. doi: 10.3389/fpls.2017.01736. eCollection 2017.
For successful infection, a virus requires various host factors at different stages such as translation, targeting, replication, and spreading. One of the host genes upregulated after infection with (BaMV), a single-stranded positive-sense RNA potexvirus, assists in viral movement. To understand how this host protein is involved in BaMV movement, we cloned its full-length cDNA by rapid amplification of cDNA ends. The gene has 3199 nt and encodes a 969-amino acid polypeptide. The sequence of the encoded polypeptide is orthologous to that of elicitor-inducible leucine-rich repeat (LRR) receptor-like protein (), a plant viral resistance gene, and is designated . To reveal how is involved in BaMV movement, we fused green fluorescent protein (GFP) to its C-terminus. Unfortunately, the gene's expression in was beyond our detection limit possibly because of its large size (∼135 kDa). However, at such low expression could still enhance BaMV accumulation in inoculated leaves. A short version of was constructed to remove the LRR domain, NbEILP/ΔLRR-GFP; the expression of this deletion mutant could still enhance BaMV accumulation to 1.7-fold that of the control. Hence, the LRR domain in NbEILP is not an essential element in BaMV movement. We constructed a few deletion mutants - NbEILP/ΔLRRΔTMD (without the transmembrane domain), NbEILP/ΔLRRΔCD (without the cytoplasmic domain), and NbEILP/ΔLRRΔSP (without the signal peptide) - to examine whether these domains are involved in BaMV movement. For BaMV movement, NbEILP requires the signal peptide to target the endoplasmic reticulum and the transmembrane domain to retain on the membrane.
为了成功感染,病毒在翻译、靶向、复制和传播等不同阶段需要多种宿主因子。感染单链正义RNA马铃薯X病毒(BaMV)后上调的宿主基因之一有助于病毒移动。为了了解这种宿主蛋白如何参与BaMV移动,我们通过cDNA末端快速扩增克隆了其全长cDNA。该基因有3199个核苷酸,编码一个969个氨基酸的多肽。编码多肽的序列与激发子诱导的富含亮氨酸重复(LRR)受体样蛋白(EILP)的序列直系同源,EILP是一种植物病毒抗性基因,该基因被命名为NbEILP。为了揭示NbEILP如何参与BaMV移动,我们将绿色荧光蛋白(GFP)融合到其C末端。不幸的是,该基因在本氏烟草中的表达超出了我们的检测限,可能是因为其分子量较大(约135 kDa)。然而,如此低表达的NbEILP仍能增强接种叶片中BaMV的积累。构建了一个去除LRR结构域的NbEILP短版本NbEILP/ΔLRR-GFP;该缺失突变体的表达仍能将BaMV积累增强至对照的1.7倍。因此,NbEILP中的LRR结构域不是BaMV移动的必需元件。我们构建了一些缺失突变体——NbEILP/ΔLRRΔTMD(没有跨膜结构域)、NbEILP/ΔLRRΔCD(没有细胞质结构域)和NbEILP/ΔLRRΔSP(没有信号肽)——以检查这些结构域是否参与BaMV移动。对于BaMV移动,NbEILP需要信号肽靶向内质网,需要跨膜结构域保留在膜上。