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Review
. 2007 Dec;145(4):1100-9.
doi: 10.1104/pp.107.106641.

New gateways to discovery

Michael M Goodin  1 , Romit Chakrabarty, Rituparna Banerjee, Sharon Yelton, Seth Debolt
Affiliations
Review

New gateways to discovery

Michael M Goodin et al. Plant Physiol. 2007 Dec.
No abstract available

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Schematic representations of pSITE vectors. A, All modified pSAT6 cassettes were cloned into the pRCS2-ocs-nptII binary vector at the PI-Psp1 site. The ability to select transgenic plant cells is conferred by the nptII gene, the expression of which is controlled by the ocs promoter (P-OCS) and terminator (T-OCS). B, C-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of proteins of interest fused to the carboxy termini of AFPs. C, N-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of proteins of interest fused to the amino termini of AFPs. D, 0-series pSITE vectors for Gateway recombination-mediated construction of binary vectors for expression of native proteins. Protein expression is controlled by a duplicated CaMV 35S promoter (2X35S) and a tobacco etch virus translational leader (TL). All vectors employ the CaMV35S transcriptional terminator (TER). Nco1*, This restriction site was deleted to create the pSITE-NB and pSITE-0B vectors, thereby allowing translation to initiate at the native start codon on the gene of interest. This figure is a revision of that appearing in Chakrabarty et al. (2007). [See online article for color version of this figure.]
Figure 2.
Figure 2.
Transient expression of AFPs from pSITE vectors. A to F, Expression of proteins whose subcellular localization cannot be determined in silico. Fluorescence from DAPI (A) and GFP (B) in the nucleus of a cell expressing a GFP:PYDV-P protein. The overlay of A and B is shown in C. Fluorescence from DAPI (D) and RFP (E) in the nucleus of a cell expressing a RFP:PYDV-N protein. The overlay of D and E is shown in F. Although both PYDV-N and -P proteins are entirely localized to the nucleus, analysis of their primary structure failed to identify karyophillic domains. G to J, Expression of AFP fusions in callus cells of N. benthamiana. G, GFP fluorescence of transgenic callus cell expressing mGFP5-ER. H, Overlay of G and I. I, RFP fluorescence following agromediated expression of RFP-SYNV-P from a pSITE vector. J, Differential interference contrast image of cell shown in G to I. K to V, Expression to study differential protein localization in pathogen-infected cells. Shown are confocal micrographs of RFP fusions of SYNV proteins expressed in SYNV-infected and mock-inoculated mGFP5-ER transgenic N. benthamiana plants. Fluorescence images for GFP, RFP, and the corresponding overlay are shown for each fusion expressed in SYNV-infected cells. Only the overlay is shown for fusions expressed in mock-inoculated leaves. Sections from top to bottom show localization of RFP:P (K–N), RFP:N (O–R), and RFP:M (S–V). Sections K to V are reprinted from Goodin et al. (2007).
Figure 3.
Figure 3.
Confocal micrographs showing localization of AFPs targeted to a variety of subcellular loci. All markers are expressed from pSITE vectors in N. benthamiana. Micrographs marked with dashed lines represent transient expression while solid lines represent stable expression in transgenic plants. Plants expressing a RFP:SYNV-M fusion were not resistant to infection. Instead, RFP:SYNV-M was relocalized from the nucleoplasm to foci consistent with sites where intranuclear membranes accumulate (see Fig. 2). Clockwise, cell marker proteins used were the Rubisco small subunit (chloroplast), soybean (Glycine max) mannosidase (Golgi), Fib1 (nucleolus), RFP-HDEL (ER), Histone 2B (chromatin), and SYNV:M protein (nuclelus). Drawing of a plant cell courtesy of http://www.ualr.edu/botany/plantcelldiagram.jpg.
Figure 4.
Figure 4.
A to F, Laser scanning confocal micrographs of N. benthamiana leaf epidermal cells showing shift in the localization of expression patterns of the RFP:AtFib1 nucleolar marker from two loci in mock-inoculated cells (A–C; mock) to three in SYNV-infected cells (D–F; SYNV). Transient expression of RFP:AtFib1 was conducted in mGFP5-ER plants. G, Quantitative comparison of RFP:AtFib1 expression patterns in mock-inoculated and SYNV-infected cells. Expression patterns were divided into two categories: nuclei with one nucleolus (1, light gray), or two nucleoli (2, dark gray). Results obtained using agroinfiltration are shown on the left while those obtained with transgenic plants are on the right. The numbers of nuclei examined (n) are shown at the bottom of the graph. Sections A to F and agroinfiltration data in G are reprinted from Chakrabarty et al. (2007).
Figure 5.
Figure 5.
A, The underlying principle of TIRFM is that incident light rays (dashed line) that strike the interface of two media of differing refractive indices (n) at greater than the critical angle (θc) result in the rays being totally internally reflected instead of passing through the second medium. At the point of reflection an evanescent wave is produced in the medium of lower refractive index. Fluorophores (green dots) entering the evanescent wave, which is in the order of 100 nm, are excited (red dots), resulting in fluorescence detection with exceedingly high signal to noise ratios. B, TIRF micrograph of an N. benthamiana protoplast expressing m5GFP-ER. C and D, TIRF micrographs showing time series of ER tubule extension and fusion. Note that the point of fusion occurs at loci where puncta form at the termini (C) or within (D) tubules. E, Confocal micrographs showing ER tubule extension and fusion lack the speed and resolution of TIRFM. F, Model suggesting that puncta form and define the sites of ER tubule fusion.
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