PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions.
Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.
Chakrabarty
Romit
R
Department of Plant Pathology, University of Kentucky, Lexington 40546, USA.
Banerjee
Rituparna
R
Chung
Sang-Min
SM
Farman
Mark
M
Citovsky
Vitaly
V
Hogenhout
Saskia A
SA
Tzfira
Tzvi
T
Goodin
Michael
M
D015153:Blotting, Western; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000821:virology; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D014780:Viruses / Q000254:growth & development*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein