Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology

Abstract

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

Keywords: CRISPR/Cas9; Potyviridae; cucumber; eIF4E; genome editing; virus resistance.

Figure 1

Gene editing of eIF4E mediated by CRISPR/Cas9 in transgenic cucumber plants. (A) Schematic representation of the cucumber eIF4E genomic map and the sgRNA1 and sgRNA2 target sites (red arrows). The target sequence is shown in red letters together with the restriction site (underlined), and the protospacer adjacent motif (PAM) is marked in bold underlined letters. The black arrows indicate the primers flanking the target sites used to detect the mutations. (B) Restriction analysis of T0 polymerase chain reaction (PCR) fragments of CEC‐1, CEC1‐4 and CEC2‐5. (C) Alignment of nine colony sequences from the undigested fragment of line 1 with the wild‐type (wt) genome sequence. DNA deletions are shown by red dashes and deletion sizes (nucleotides) are marked on the right side of the sequence.

Figure 2

Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1‐1 line. (A) Polymerase chain reaction (PCR) restriction analysis of Cas9/sgRNA1‐mediated mutations (top panel) and transgene insertion (bottom panel) in 10 representative T1 cucumber plants and non‐mutant wild‐type (wt). (B) Alignment of four representative eif4e mutant plants with the wild‐type sequence. The sequences of each plant represented clones from undigested fragments. The target sequence is shown in red letters and the protospacer adjacent motif (PAM) is marked by bold underlined letters. DNA deletions are marked with red dashes and deletion sizes (nucleotides) are indicated on the right side of the sequence.

Figure 3

Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1‐4 line. (A) Polymerase chain reaction (PCR) restriction analysis of Cas9/sgRNA1‐mediated mutations (top panel) and transgene insertion (bottom panel) in eight T1 cucumber plants and non‐mutant wild‐type (wt). (B) Alignment of three eif4e transgenic mutant plants 4, 5 and 6 with the wild‐type sequence. Sequences of each plant represent clones from undigested fragments. The target sequence is shown in red letters and the protospacer adjacent motif (PAM) is marked in bold underlined letters. DNA deletions are marked by red dashes and deletion sizes (nucleotides) are indicated on the right side of the sequence.

Figure 4

Genotyping of the Cas9/sgRNA2‐mediated mutation in T1 progeny plants of the CEC2‐5 line. (A) Polymerase chain reaction (PCR) restriction analysis of Cas9/sgRNA2‐mediated mutations (top panel) and the presence of the Cas9/sgRNA2 transgene (bottom panel) in eight representative T1 cucumber plants. (B) Alignment of four representative eif4e mutant plants with the wild‐type sequence. The target sequence is shown in red letters and the protospacer adjacent motif (PAM) is marked in bold underlined letters. DNA deletions or insertions are marked by red dashes and letters, and the sizes of the deletions or insertions (nucleotides) are indicated on the right side of the sequence.

Figure 5

Homozygous eif4e mutant plants exhibited immunity to Cucumber vein yellowing virus (CVYV) infection. (A) Disease symptoms (leaves and plants) of heterozygous (Het‐mut), homozygous (Hom‐mut) and non‐inoculated (Control) plants of the CEC1‐1‐7‐1 T3 generation at 10 days post‐infection (dpi). (B) Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of CVYV RNA accumulation at 14 dpi in homozygous eif4e mutant plants (plants 1–11), heterozygous eif4e mutant plant (Het.) and non‐inoculated plant (Control). TIP41 (tonoplast intrinsic protein) was used as a reference gene for RT‐PCR amplification. A molecular marker 100‐bp ladder is shown (M).

Figure 6

Homozygous eif4e mutant plants exhibited resistance to Zucchini yellow mosaic virus (ZYMV) infection. (A) Disease symptoms of heterozygous (Het‐mut), homozygous (Hom‐mut) and non‐inoculated (Control) plants of the CEC1‐1‐7‐1 T3 generation at 25 days post‐infection (dpi). (B) Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of ZYMV RNA accumulation in homozygous eif4e mutant plants (1–10), heterozygous plants (Het‐mut) and non‐inoculated plant (H) at 14 dpi. Tip41 was used as a reference gene for RT‐PCR amplification. A molecular marker 100‐bp ladder is shown (M). (C) Relative (real‐time quantitative RT‐PCR) ZYMV RNA accumulation in CEC1‐1‐7‐1 heterozygous (Het‐mut) and two classes of homozygous mutant: resistant (Resistant) and breaking (Break). RNA was extracted from three plants (third top leaf) and the ZYMV level was calculated using the ΔΔCT method normalized to the F‐box gene expression level.

Figure 7

Homozygous eif4e mutants exhibited resistance to Papaya ring spot mosaic virus‐W (PRSV‐W) infection. (A) Disease symptoms of heterozygous (Het‐mut), homozygous (Hom‐mut) and non‐inoculated (Control) plants of CEC1‐1‐7‐1 T3 generation at 21 days post‐infection (dpi). (B) Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of PRSV‐W RNA accumulation in homozygous plants (1–8), heterozygous plant (Het.) and non‐inoculated plant (H) at 14 dpi. Tip41 was used as a reference gene for RT‐PCR amplification. A molecular marker 100‐bp ladder is shown (M). (C) Relative (real‐time quantitative RT‐PCR) accumulation of PRSV‐W RNA in CEC2‐5‐M‐9 heterozygous (Het‐mut) and three classes of homozygous mutant: resistant (Resistant), breaking (Break) and recovering (Recovery). RNA was extracted from the second top leaf of three plants and the PRSV‐W RNA level was calculated using the ΔΔCT method normalized to the F‐box gene expression level.