New Gateways to Discovery 1(C)

Michael M. Goodin*, Romit Chakrabarty, Rituparna Banerjee, Sharon Yelton, and Seth DeBoltDepartment of Plant Pathology (M.M.G., R.C., R.B., S.Y.) and Department of Horticulture (S.D.), University ofKentucky, Lexington, Kentucky 40546Dr.Panglosswasright,atleastforlive-cellimaginginplants, in his contention that this is ‘‘the best of allpossible worlds’’ (Voltaire, 1759). We are now able tolook inside cells in detailed ways that the fathers ofmicroscopy, Antoni van Leeuwenhoek and RobertHook, could not have possibly imagined (Gest, 2004).Current state-of-the-art instrumentation has made rou-tine an array of imaging techniques that were inac-cessible to most researchers just a few years ago.Additionally, the number of autofluorescent proteins(AFPs) with which we can color intercellular structuresincreases almost daily, thus providing ever greaterflexibility in the types of experiments that can beperformed. Moreover, the completion of large-scalegenome sequencing projects has sparked many deriv-ative microscopy-based projects, which are essential toputthefunctionintofunctionalgenomicsandtorealizetheemergentfieldofsystemsbiology(Murphy,2005;Lietal.,2006;PepperkokandEllenberg,2006).Forindeed,if a picture is still worth a thousand words (Orenstein,2000), consider all that will be learned when, by mi-croscopy en masse, we visualize the thousands ofproteins predicted to be encoded in the genomes ofeukaryotes in general (Brasch et al., 2004; Rual et al.,2004; Matsuyama et al., 2006) and plants in particular(Sterck et al., 2007).This review is designed primarily to introduce re-searchers to the myriad of factors, beyond vectorselection, that should be considered prior to embark-ing on protein localization experiments (Table I). Tobegin, we will review some of the most recently pub-lished vector systems for the expression of AFPs inplant cells, with particular emphasis on the pSAT(modular satellite plasmid) and pSITE (stable integra-tion and transient expression plasmid) vectors (Tzfiraet al., 2005; Chakrabarty et al., 2007; Goodin et al.,2007). Using the pSITE vectors, we have developedseveral transgenic lines to support cell biology studiesconducted with Nicotiana benthamiana, a model hostessential for the study of plant-pathogen interactions,which is being utilized increasingly in plant biologyresearch, primarily due to the facile manner by whichlarge populations of cells can be transfected. Next, wewill compare protein localization data produced usingtransient assays versus transgenic plants. We will alsodiscuss recent results from our lab supporting thatprotein localization data obtained using transientassays is comparable to that obtained from transgenicplants (Chakrabarty et al., 2007; Goodin et al., 2007).Finally, we discuss how advances in AFP-vector de-velopment will realize their greatest utility when usedin conjunction with state-of-the-art imaging systems.Therefore, we conclude this review by examining theapplication of total internal reflectance fluorescencemicroscopy (TIRFM) as an adjunct to confocal imagingwith a study of endoplasmic reticulum (ER) dynamicsas an example.