Mediterranean Agronomic Institute of Chania, Department of Sustainable Agriculture, Alsylio Agrokepio, GR-73100 Chania, Greece.
Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida, 25198 Lleida, Spain.
Int J Mol Sci. 2018 Nov 25;19(12):3747. doi: 10.3390/ijms19123747.
(PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (TXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. TXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy plants, TXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-TXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 pellets, and the soluble fraction of extracts of healthy and PepMV-infected leaves, but not in the cell wall. These bands likely consist of the homologous protein TXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected leaves using α-TXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.
（ PepMV ）是一种在世界范围内具有重要意义的机械传播番茄病原体。我们实验室之前曾报道过 PepMV 外壳蛋白与三基因块蛋白（ TGBp1 ）分别与宿主热休克同源蛋白 70 和过氧化氢酶 1 （ CAT1 ）相互作用。在这项研究中，通过酵母双杂交筛选、体外下拉和双分子荧光互补（ BiFC ）实验，发现了一种新的番茄互作蛋白（ TXND9 ）与 PepMV TGBp1 结合。TXND9 具有部分保守的硫氧还蛋白（ TRX ）活性位点序列（ W__PC 与 WCXPC ），并且 TXND9 同源物在 TRX 系统发育超家族中与磷蛋白 3 最接近。在 PepMV 感染和健康植株中， TXND9 mRNA 水平相当，并且在接种后 10 天内，局部和系统叶片中的表达水平保持稳定，过氧化氢酶 1 （ CAT1 ）也是如此。为了在植物细胞中定位 TXND9 ，制备了多克隆抗血清。纯化的α-TXND9 免疫球蛋白（ IgG ）在健康和 PepMV 感染的 叶片提取物的 1000 和 30000 沉淀以及可溶性部分中，一致地检测到一组三个蛋白条带，范围为 27⁻35 kDa ，但不在细胞壁中。这些条带可能由同源蛋白 TXND9 及其翻译后修饰衍生物组成。在电子显微镜下，使用α-TXND9 IgG 对 PepMV 感染的 叶片超薄切片进行免疫金标记，发现颗粒在质膜附近聚集，提示其在病毒运动中起作用。综上所述，这项研究强调了番茄与 PepMV 之间的一种新的蛋白相互作用，并提供了其在植物体内定位的相关数据。目前，正在进行专注于 PepMV 感染期间这种相互作用的生物学功能的研究。
