Development of super-infective ternary vector systems for enhancing the Agrobacterium-mediated plant transformation and genome editing efficiency

Abstract

Agrobacterium-mediated transformation remains a cornerstone of plant biology, fueling advancements in molecular genetics, new genomic techniques (NGTs), and the biotech industry. However, recalcitrant crops and technical hurdles persist as bottlenecks. The goal was to develop super-infective ternary vector systems that integrate a novel salicylic acid-degrading enzyme, GABA, and ethylene-degrading enzymes, targeting the transformation of crops by neutralizing plant defense system on Agrobacterium. Firstly, both the effect and activity of introducing enzymes were validated in EHA105, an important Agrobacterium strain. Our study demonstrates that all ternary vector (Tv) system variants significantly enhance reporter expression in transient assays with Nicotiana benthamiana and Cannabis sativa. Specifically, incorporating a constitutive virG mutation with novel enzyme combinations increased GFP and RUBY expression in C. sativa by >5-fold and 13-fold, respectively. The Tv system, combined with a geminivirus replicon, markedly boosted GUS gene expression in tomato, enhancing genome editing efficiency. Notably, compared to controls, Tv-VS demonstrated up to 18-fold and 4.5-fold increases in genome editing efficiency in C. sativa and tomato, respectively. Additionally, stable transformation rates in tomato and Arabidopsis improved significantly, with Tv-VS showing a remarkable 2.5-fold increase in transformation efficiency compared to control strains. The research marks notable progress in Agrobacterium-mediated plant transformation. The innovative ternary vectors overcome plant defense mechanisms, enabling genetic manipulation in previously challenging plant species. This development is anticipated to broaden the applications of plant genetic engineering, contributing to advancements in crop genome editing.

Figure 1

Validation of ACC deaminase, GABA transaminase, and salicylate hydroxylase in A. tumefaciens.  (A) Effect of ternary vectors on Agrobacterium growth. (B) Comparison of Agrobacterium growth at the end of the exponential stage (40 h). The enzymatic reaction of ACC deaminase (C), GABA transaminase activity (E), and salicylate hydroxylase activity (G). Confirmation of ACC deaminase activity, which measured production of α-ketobutyrate in cell lysates (D), GABA transaminase activity, which measured glutamate production in cell lysates (F), and salicylate hydroxylase activity, which measured decrease in salicylate absorbance in cell lysates (H). Data are from at least three independent experiments with biological replicates and are expressed as means ± SD. Different characters indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with P < 0.05.

Figure 2

Transient transformation assay in C. sativa.  (A) Visualization of Clover GFP expression in C. sativa leaf using Azure 600 Imaging system.  (B) Representative western blot analysis with anti-GFP antibodies was performed in C. sativa leaf. Actin served as loading control. (C) Quantification of GFP protein levels in C. sativa using ImageJ. Data are from at least three independent experiments with biological replicates and are expressed as means ± SD. These plants were incubated for 4 days after agro-infiltration using Agrobacterium strains carrying GFP construct. The visible RUBY expression (D) and extraction of betalain pigments (E) from RUBY-transformed Cannabis-immature embryos. (F) Betalain contents were measured spectrophotometrically in extracts of Cannabis embryos. Data are from at least three independent experiments with biological replicates and are expressed as means ± SD. These embryos were incubated for 3 days after co-cultivation using Agrobacterium strains carrying RUBY construct. Different characters indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with P < 0.05.

Figure 3

Evaluation of T-DNA transfer frequency with geminivirus-based replicon system in Tv system. GUS expression cassettes in T-DNA region of the geminivirus-based replicon (A) and T-DNA based conventional binary vector (B). (C) GUS-stained explants of tomato cotyledons after Agrobacterium-mediated transient transformation by the geminivirus-based replicon. (D) The percentage of GUS-stained area was evaluated in tomato cotyledons by conventional T-DNA based binary vector and geminivirus-based replicon. Values are means ± SD (n = 36). Different characters indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with P < 0.05. Asterisks indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. (E) Comparison of the GUS-stained percentage of each strain in the presence or absence of AS. Values are means ± SD (n = 36). Asterisks indicate a statistically significant difference based on unpaired t-test, with ^*P < 0.05 and ^***P < 0.001. Agrobacterium strains contained GUS-construct. Scale bars in images represent 1 mm.

Figure 4

Synergistically increased efficiency of genome editing via Tv system and geminiviral replicon in C. sativa.  (A) Schematic representation of CRISPR/Cas9 expression with CsTHCAS-target gRNAs in T-DNA region of geminivirus-based replicon. Human-optimized SpCas9 (hCas9) and CsTHCAS-target gRNAs were driven by 35S promoter with a duplicated enhancer (p2x35S) and AtU6 promoters, respectively. (B) Schematic map of the target gene (CsTHCAS). Thin vertical lines represent the target sites within the coding sequence (CDS). The letters below the bar indicate target sequences. Indel frequencies for CsTHCAS-gRNA1 (C) and -gRNA2 (D) in Cannabis-immature embryos determined by targeted deep sequencing. Data are from at least three independent experiments with biological replicates and are expressed as means ± SD. Different characters indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with P < 0.05. Agrobacterium strains contained Cas9-THCAS-construct in replicon.

Figure 5

Improvement of genome editing, stable transformation, and regeneration via Tv system. (A) Schematic representation of CRISPR/Cas9 expression in T-DNA. 35S with a duplicated enhancer (p2x35S) and AtU6 promoters were used for hCas9 and gRNA, respectively. Red arrows indicate a set of specific primers for T-DNA insertion. Schematic maps of the target genes SlAnt1  (B) and SlPsy1  (C). Thin vertical lines represent the target site within the coding sequence (CDS). The letters below the bar indicate target sequences. Indel frequencies for SlAnt1  (D) and SlPsy1  (E) in tomato cotyledons determined by targeted deep sequencing. (F) Kanamycin-resistant shoots were regenerated from tomato cotyledons with Agrobacterium strains carrying the Cas9 construct. Scale bars, 1 cm. Effect of Tv system on shoot regeneration (G), transformation efficiency (H), and copy number of T-DNA (I). (J) Genome editing efficiency was determined by analyzing T-DNA-inserted T₀ plants for edits in the targeted gene (SlAnt1), and the editing was confirmed through Sanger sequencing. Data are from at least three independent experiments with biological replicates and are expressed as means ± SD. Different characters indicate a statistically significant difference based on one-way ANOVA and Tukey's multiple range test, with P < 0.05. Agrobacterium strains contained Cas9-(Ant1 or Psy1)-construct.

Figure 6

Schematic representation of super-infective ternary vector system. Agrobacterium perceives plant signals, such as neutral sugars, phenolic compounds, and an acidic pH, leading to the induction of Agrobacterium virulence gene expression. In contrast, Vir gene expression is inhibited by negative factors, such as ethylene, IAA, SA, and GABA. VIR proteins play a crucial role in the T-DNA transfer processes facilitated by the type IV secretion system. The successful integration leads to the expression of the T-DNA-encoded genes. In the super-infective ternary vector system, a third plasmid is introduced, carrying the host defense-avoidance mechanisms, and promoting Vir gene induction, thus dramatically increasing the transformation efficiency.