Howard Amanda R, Heppler Marty L, Ju Ho-Jong, Krishnamurthy Konduru, Payton Mark E, Verchot-Lubicz Jeanmarie
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Virology. 2004 Oct 25;328(2):185-97. doi: 10.1016/j.virol.2004.06.039.
Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereas GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.
开展了实验以比较马铃薯X病毒(PVX)的三基因块蛋白1(TGBp1)和外壳蛋白(CP)在几种植物物种中的胞间连丝转运活性。显微注射实验表明,TGBp1可打开烟草叶片中的胞间连丝。这些结果支持了之前的显微注射研究,该研究表明TGBp1可打开本氏烟草和克利夫兰烟草叶片中的胞间连丝。为了研究蛋白质的移动,将与PVX TGBp1或CP基因融合的绿色荧光蛋白(GFP)基因表达质粒通过生物弹道技术轰击取自四种不同PVX宿主物种的叶片。GFP/TGBp1可在烟草、克利夫兰烟草、本氏烟草和番茄的相邻细胞间移动,而GFP/CP仅在本氏烟草叶片中移动。将m12和m13突变引入TGBp1基因,这两种突变均消除了TGBp1的ATP酶活性位点基序,抑制了PVX的移动,减少了GFP/TGBp1在本氏烟草叶片中的细胞间移动,并消除了GFP/TGBp1在烟草、克利夫兰烟草和番茄叶片中的移动。GFP/TGBp1m13在烟草细胞中形成聚集体。GFP/CP和突变型GFP/TGBp1融合蛋白在本氏烟草中能够移动而在其他PVX宿主物种中不能移动,这表明本氏烟草植株具有促进蛋白质细胞间移动的独特能力。