Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector

Abstract

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS-P system to investigate the expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti-HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct.

Keywords: Griffithsin; MIDAS-P; VRC01; anti-HIV cocktail; modular; tandem plant expression vector.

Figure 1

Schematic representation of the MIDAS‐P assembly system for plant expression. The system consists of two entry vectors, pWHITE and pBLUE, for cloning genes of interest and alternate sub‐cloning in the binary destination (expression) vector pMIDAS. The first transcriptional unit is constructed in pWHITE and transferred into pMIDAS using the type IIS restriction enzyme BsaI. A second transcriptional unit in pBLUE can subsequently be transferred into pMIDAS using BsmBI. Further TUs can be added by alternating transfer from pWHITE and pBLUE. The inclusion of lacZα in pMIDAS and pBLUE allows blue/white screening at each stage. The destination vector pMIDAS also has right and left T‐DNA borders for Agrobacterium‐mediated plant transformation. GOI, gene of interest; P, promoter; pA, terminator and polyA signals; SAR, scaffold attachment region; UTR, untranslated region

Figure 2

DsRed expression level with increasing number of TUs. (a) The arrangement of the individual transcription units in tandem in pMIDAS. DsRed (red box) was cloned into the last position in constructs harboring 2–5 TUs. The other TUs can include VRC01 heavy chain (HC; dark green), VRC01 light chain (LC; blue), Griffithsin (GRFT; light green) and 6x‐His tagged J3‐VHH (J3HS; yellow). (b) DsRed expression levels in leaves infiltrated with 1 TU (DsRed), 2 TUs (HC‐DsRed), 3 TUs (HC‐LC‐DsRed), 4 TUs (HC‐LC‐GRFT‐DsRed), or 5 TUs (HC‐LC‐GRFT‐J3His‐DsRed), as well as leaves co‐infiltrated with 4 TUs and DsRed as separate constructs (DsRed with HC‐LC‐GRFT‐J3His), quantified at 6 dpi. Leaves infiltrated with pMIDAS only were used as a negative control (Control). Box plot for DsRed expression levels represent the mean, minimum, and maximum of six biological repeats. Data were analyzed using Brown–Forsythe and Welch ANOVA tests with Tamhane T2 multiple comparison test (*p < 0.033 and **p < 0.002). ANOVA, analysis of variance; TUs, transcription units

Figure 3

Positional effects with TU permutations on protein expression of MIDAS‐P constructs. Representative western blots of extracts from leaves infiltrated with different constructs harboring permutations of 3 TUs (a) and of 4–5 TUs (b) at 6 dpi. HC‐LC‐GRFT‐J3His is shown as a representative example of 4 TU constructs. Leaves infiltrated with pMIDAS were used as negative controls (−ve control). Positive controls (+ve control) were leaves infiltrated with either a 2 TU construct containing VRC01 HC + LC, or a 1 TU construct containing GRFT or 6x histidine tagged J3‐VHH (J3His). (c) DsRed expression levels in 4 or 5 TU constructs. Leaves infiltrated with pMIDAS only were used as negative controls (Control). Expression levels were quantified using a DsRed standard and box plots represent the mean, minimum and maximum of six biological repeats. Data were analyzed using Brown‐Forsythe and Welch ANOVA tests with Tamhane T2 multiple comparison test (***p < 0.001 and ****p < 0.0001). ANOVA, analysis of variance; TUs, transcription units

Figure 4

Quantification of mRNA levels of Nicotiana benthamiana leaves infiltrated with MIDAS‐P constructs harboring different combinations of transcription units. (a) Positive controls showing mRNA levels in leaves infiltrated with MIDAS‐P constructs carrying two TUs for VRC01 heavy chain (HC) and light chain (LC), or a single TU for expression of Griffithsin (GRFT), 6xHis‐tagged J3‐VHH (J3His) or DsRed (b–d) VRC01 HC (b), LC (c) and GRFT (d) mRNA levels in leaves infiltrated with MIDAS‐P constructs with different permutations of 3 TUs expressing VRC01 HC, LC, and GRFT; or with 4 TUs (additional TU expressing J3His), compared with the respective positive controls on Figure (a) (J3His data not shown). Data for (b), (c), and (d) were analyzed using Brown‐Forsythe and Welch ANOVA tests followed by Tamhane T2 multiple comparison test (****p < 0.0001; **p < 0.01, *p < 0.05). (e–i) VRC01 HC (e), LC (f), GRFT (g), J3His (h), and DsRed (i) mRNA levels in leaves infiltrated with MIDAS‐P constructs with different permutations of 5 TUs expressing the 5 genes and a repeat of the 4 TU construct from (b–d), compared with the respective positive controls on Figure (a). Please refer to Figure 4i for x‐axis labels of e–h. Data for (e), (g), and (i) were analyzed using Brown‐Forsythe and Welch ANOVA tests with Tamhane T2 multiple comparison test; (f) and (h) were analyzed using Dunn Bonferroni multiple comparison test (LC: Kruskal–Wallis test statistic = 6.7; J3His vs. HC‐LC‐GRFT‐J3His p < 0.0001, Kruskal–Wallis test statistic = 40.2). Leaves from plants infiltrated with infiltration solution served as negative control (“Control”). mRNA levels were determined with qPCR using the primers listed in Table S2. All data represent the mean of three biological repeats ± SD. ANOVA, analysis of variance; mRNA, messenger RNA; TUs, transcription units; SD, standard deviation

Figure 5

Coexpression of P19 with constructs harboring 3 or 5 TUs. (a) mRNA levels of leaves co‐infiltrated with the pMIDAS construct with 3 TUs for expression of the VRC01 heavy chain (HC), light chain (LC), and Griffithsin (GRFT) with pMIDAS‐P19 were determined by qPCR using the primers listed in Table S2. Data represent the mean of n = 3 biological repeats done in triplicates ± SD. Data were analyzed using Brown–Forsythe and Welch ANOVA tests followed by Tamhane T2 multiple comparison test (***p < 0.001 and ****p < 0.0001). (b) Protein expression levels of VRC01 and GRFT, 6x His‐tagged J3‐VHH (J3His) and DsRed expressed using 3 TU and (c) 5 TU constructs with or without P19 co‐expression. VRC01, GRFT, J3‐VHH, and DsRed expression levels were quantified by ELISA using a human IgGk standard (Sigma‐Aldrich), GRFT standard (gift from Evangelia Vamvaka and Paul Christou), plant‐made J3‐VHH and DsRed standard (gift from Johannes Buyel) respectively. Box plot for expression levels represent the median, minimum, and maximum of three biological repeats. ANOVA, analysis of variance; ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA; TUs, transcription units; SD, standard deviation