Tcs sp2 inverted confocal microscope

Construction of GmDGAT1A- and 2D-GFP fusion was done by using gateway recombination into pK7WGF2 in fusion with N-terminus of GFP. Determination of the subcellular localization of GmDGAT-GFP was performed by using tobacco leaf infiltration following the method described previously50 (link). GmDGAT-GFP and ER marker CD3-959: Cherry DNAs were transformed into Agrobacterium strain K599, which were used for plant transformation. The acetosyringone-activated bacteria were co-infiltrated into the abaxial epidermal surface of a tobacco leaf with a syringe, and the plant was grown at room temperature for 2 to 3 d before imaging. Imaging of GmDGAT-GFP fusion proteins was performed using a Leica TCS SP2 inverted confocal microscope using a 63×water-immersion objective and Leica Confocal software with an excitation wavelength of 488 nm and emissions collected at 500 nm. CD3-959-mCherry–labeled ER membrane was excited at 543 nm with the argon laser, and emission was detected from 620 to 680 nm. Chloroplasts were visualized at 560 to 610 nm under excitation at 543 nm.

The subcellular localization of GmCYP1 was studied by infiltrating A. tumefaciens GV3101 carrying pEG101-GmCYP1 into N. benthamiana leaves, as described by Sparkes et al.67 . For co-expression, equal volumes of two construct-bearing strains, suspended in Gamborg’s solution, were mixed together and then infiltrated into N. benthamiana leaf epidermal cells. The protein expression was visualized by confocal microscopy using a Leica TCS SP2 inverted confocal microscope. An excitation wavelength of 514 nm was used for YFP imaging, and 525–545 nm emissions were collected. For visualization of CFP, an excitation wavelength of 458 nm was used, and emissions were collected between 465–495 nm.

For subcellular localization study, GmCHSs were amplified from soybean cDNA by PCR using gene-specific primers. Primers used for GmCHSs amplification are listed in Additional file 6: Table S6. The PCR products were cloned into the gateway entry vector pDONR-Zeo (Invitrogen) using BP clonase (Invitrogen), followed by transformation into Escherichia coli DH5α. The recombinant plasmid pDONZ-GmCHS was sequence confirmed and recombined with the destination vector pEGmCherry101using LR clonase reaction mix (Invitrogen). The recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 via electroporation. To create pEGmCherry101, mCherry fragment was amplified by PCR using primers AvrII-mCherry-F and XbaI-6His-mCherry-R (Additional file 6: Table S6). The resulting PCR products were digested with AvrII and XbaI, and inserted into the AvrII site at the N-terminus of the YFP in pEarleyGate101 [42 (link)].
The pEGmCherry-GmCHS constructs in A. tumefaciens GV3101 were transformed into Nicotiana benthamiana leaf by infiltration [43 (link)] and transient expression was visualized through a Leica TCS SP2 inverted confocal microscope. For confocal microscopy, a 63X water-immersion objective was used at excitation wavelengths at 514 nm and emission spectra of 530-560 nm for YFP.

For subcellular localization study, GmCHRs were amplified by PCR using gene-specific primers (Supplementary Table S1) and cloned into the gateway entry vector pDONR-Zeo (Invitrogen) using the BP clonase reaction mix (Invitrogen), transformed into Escherichia coli DH5α. The recombinant plasmid pDONR-Zeo-GmCHR was recombined with the destination vector pEarlyGate101 using the LR clonase reaction mix (Invitrogen). The sequence confirmed pEarlyGate101 was transformed into Agrobacterium tumefaciens GV3101 via electroporation.
The constructs in A. tumefaciens GV3101 were transformed into N. benthamiana leaves by infiltration (Sparkes et al., 2006 (link)), and transient expression was visualized through a Leica TCS SP2 inverted confocal microscope. To confirm the nuclear localization of the proteins, A. tumefaciens GV3101 with the construct containing nuclear localization signal fused to CFP (pEarleyGate100-NLS-CFP) and pEarleyGate101-GmCHR were mixed 1:1 and infiltrated into the leaves followed by confocal microscopy. For YFP visualization, an excitation wavelength of 514 nm was used and emissions were collected between 525 and 545 nm. For visualization of CFP, an excitation wavelength of 434 nm was used and emissions were collected between 460 and 490 nm.

Confocal microscopy was performed essentially as described previously59 (link)60 (link)61 (link). Fluorescence was visualized at 2–4 dpa using a Leica TCS SP2 inverted confocal microscope (http://www.leica.com/) with an Argon-Krypton laser. Sections from agroinfiltrated leaves were excised and placed between two microscope cover slides with a drop of water. YFP signals were imaged using a 63× water immersion objective at an excitation wavelength of 514 nm, and emissions were collected between 525 and 575 nm. Images of CFP fluorescence were obtained using the same microscope at an excitation wavelength of 458 nm and emissions were collected between 470 and 500 nm. GFP signal was excited at 488 nm and the emitted light was captured at 505 to 525 nm. mCherry fluorescence was excited at 543 nm and the emitted light was captured at 590–630 nm. Light emitted at 630–680 nm was used to record chlorophyll autofluorescence. Data for the different color channels were collected simultaneously. The samples were scanned at a resolution of 512 × 512 pixels. Images were collected with a charge-coupled device camera and analyzed by Leica confocal software.

Equal volumes of two construct-bearing strains containing YFP and CFP fusions (in Gamborg’s solution), were mixed together and then infiltrated into N. benthamiana leaf epidermal cells. The protein expression was visualized by confocal microscopy using a Leica TCS SP2 inverted confocal microscope. An excitation wavelength of 458 nm and 514 nm were used for CFP and YFP imaging respectively. FRET acceptor bleaching, with CFP as donor and YFP as acceptor, was carried out by following Leica confocal application manual. The average of the FRET efficiency was calculated from multiple samples (n > 15).

The leaves of 4–6 week old N. benthamiana plants were infiltrated with A. tumefaciens culture containing GOI in the appropriate destination vectors^{54 (link)}. To verify PPI, constructs in pEarleyGate201-geneA and pEarleyGate202-geneB were co-transformed in a 1:1 mixture. Confocal microscopy was carried out 48 h post infiltration using a Leica TCS SP2 inverted confocal microscope. The excitation and emission of YFP were 514 nm and 530–560 nm, respectively.