Fig. 2: Comparison of three discrete methods to deliver normalizing reporters.

A Left, categories of delivery methods: unnormalized (green or magenta), co-infiltration (yellow), same T-DNA (pink), and BiBi (blue). Center, kernel density estimation plots of eGFP and mCherry fluorescence, n = 96 leaf disks per row. Right, cartoons showing the binary vector origin of replication, resistance marker, and orientations of FP expression cassettes in the T-DNA. All binary vector cartoons are read from left to right: ori, left border, T-DNA, right border. Origins of replication are pVS1 (diamond, black), BBR1 (circle, teal), and pSa (square, gray). Circles enclosing an X represent tOcs, a 722 bp spacer in between the two expression cassettes. B Plant coefficients of variation (CV), as calculated from the 8 disks per plant, when eGFP is treated as the reporter. All values are eGFP/mCherry CV except for scheme 1, which is GFP CV. C Plant CVs when mCherry is treated as the reporter. All values are mCherry/eGFP CV except for scheme 2, which is mCherry CV. Normalization scheme IDs match across all subpanels. Boxes show the median and IQR, and whiskers show the minima and maxima, excluding outliers (beyond 1.5 IQR). For B, C, an independent, one-tailed Welch’s t-test and a Bonferroni correction were conducted to determine whether the reporter/normalizer CV of a scheme is significantly less than the reporter CV for the corresponding unnormalized scheme. Two experimental replicates of six plants are shown in (B, C), for a total of n = 12 plants. Asterisks indicate p-values: ^*<0.05, ^**<0.01. Total OD infiltrated in all schemes is 0.5. ODs of co-infiltrated strains are 0.25 each. In the legend, “&” indicates co-infiltration, arrows indicate the direction of an expression cassette, “+” indicates BiBi, and GFP is abbreviated to “G” and mCherry to “R”. Leaves T4 and T5 of six four-week-old N. benthamiana plants were infiltrated in both experimental replicates, and four disks were collected from each infiltrated leaf. Created in BioRender. Tang, S. (2025) https://BioRender.com/0573uvz. Source data for this figure is available in the Source Data file.