Lsm900 confocal microscope imaging system

The SPX-MFS3 amino acid sequences of 19 different species were downloaded from NCBI for evolutionary analysis, and a multiple sequence alignment was constructed. The CoSPX-MFS3 coding sequences were fused with the mGFP-encoding sequences in the pMDC43 expression vector with the CloneExpress II One Step Cloning Kit (Vazyme, Nanjing, China). For transient expression analysis, the recombinant plasmids were incorporated into Agrobacterium tumefaciens EHA105 cells, prior to transfer to Nicotiana benthamiana leaves. The empty vectors were employed as controls. After three days, the transient expression of GFP-fusion proteins was determined using an LSM900 confocal microscope imaging system (Zeiss, Shanghai, China). mCherry-labeled vacuole markers were used to visualize the tonoplast.

To analyze the subcellular localization of candidate genes (DlTCP5-C, DlTCP7-B, DlTCP9-A, DlTCP12-C, and DlTCP23-C), we designed specific primers to amplify the full-length coding sequence of candidate TCP genes and then inserted them into mGFP fusion expression vector pMDC43 (Supplementary Table S2). Then, the location signal was analyzed in the leaf tissue of Nicotiana benthamiana after transforming Agrobacterium tumefaciens GV3101 as described in the previous research (Sparkes et al., 2006 (link)). The empty vector was used as the control. About 72 h later, the transient expression of GFP fusion protein was observed by LSM900 confocal microscope imaging system (Zeiss, Germany). The nucleus was visualized with mCherry-labeled nuclear markers. Subsequently, the candidate TCP genes were inserted into the pGBKT7 vector to study the transcriptional activity of DlTCP proteins in yeast (Supplementary Table S2). Then, the above recombinant vector, positive control pGBKT7-p53 + pGADT7-T, and negative control pGBKT7 empty plasmid were transformed with lithium acetate method into yeast strain AH109. The transformed strains were cultured on SD medium lacking Trp (SD-Trp) and further selected on defective medium SD-Trp-His-Ade supplemented with X-α-gal for 3–5 days. The transcriptional activation activity was evaluated according to the growth status.

To clarify the subcellular localization of the PmNBS-LRR97, the full-length sequence of the gene was ligated to the pCambia1300 expression vector with GFP at its C-terminus, and this then transformed into Agrobacterium GV3101. Using the empty vector as a control, the Agrobacterium was suspended to OD₆₀₀ = 1.2, incubated for 2 h, and then injected into the lower epidermis of tobacco leaves. Two days later, the transient expression of the GFP fusion protein was observed by the LSM900 confocal microscope imaging system (Zeiss, Oberkochen, Germany). Nicotiana benthamiana Domin was cultured in a light incubator at 25 °C, under a 16 h light/8 h dark cycle.